microM Nifedipine Research Articles

Conditioned medium from activated spleen cells supports the survival of rat retinal cells in vitro.

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 microM nifedipine. However, the use of an intracellular calcium chelator (15.0 microM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions.

Cl- and K+ conductances activated by cell swelling in primary cultures of rabbit distal bright convoluted tubules.

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl- currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl- currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I- = Br- > Cl- >> glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid and 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+ but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 microM LaCl3, 10 microM nifedipine, 100 microM verapamil, 5 microM tamoxifen, and 50 microM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+ was without effect on the fully developed Cl- currents. Osmotic shock also activated K+ currents in Cl- free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl- and K+ currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.

Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage-sensitive calcium channels.

1. Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[3H]-arginine to L-[3H]-citrulline. 2. Elevation of extracellular K+ to 20, 35 or 60 mM concentration-dependently augmented L-[3H]-citrulline production. The response to 35 mM KCl was abolished by N(G)-nitro-L-arginine (100 microM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl2 concentration up to 10 mM also prevented the K+ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity. 3. However, the effect of high K+ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)-quinoxaline-2-3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine. 4. The 35 mM K+ response was insensitive to omega-conotoxin GVIA (1 microM) and nifedipine (100 microM), but could be prevented in part by omega-agatoxin IVA (0.1 and 1 microM). The inhibition caused by 0.1 microM omega-agatoxin IVA (approximately 30%) was enhanced by adding omega-conotoxin GVIA (1 microM) or nifedipine (100 microM). Further inhibition (up to above 70%) could be observed when the three Ca2+ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 microM) the K+-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 microM omega-conotoxin GVIA plus 100 microM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX. 5. It was concluded that elevation of extracellular [K+] causes NO synthase activation by external Ca2+ entering cells mainly through channels of the P/Q-type. Other Ca2+ channels (L- and N-type) appear to contribute when P/Q-channels are blocked.

Effects of stimulation and inhibition of protein kinase C on the cytosolic calcium concentration in rabbit afferent arterioles.

The effect of the protein kinase C (PKC) inhibitor chelerytrine (Ch) and the PKC activator 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on the cytosolic calcium concentration ([Ca2+]i) in isolated intact rabbit afferent arterioles was investigated. [Ca2+]i was measured in the proximal and distal parts of the arteriole. Administration of 1 microM Ch gave rise to a peak followed by an elevated level of [Ca2+]i in both these parts. Neither the peak nor the elevated level of [Ca2+]i was significantly reduced by 1 microM nifedipine. The relative peak increase in [Ca2+]i in response to 1 microM noradrenaline (NA) or to 10 nM angiotensin II (AII) was significantly blunted in both parts after preincubation with 1 microM Ch. Depolarization with 25 mM K+ increased [Ca2+]i in both parts. Preincubation with Ch did not affect the increase in [Ca2+]i induced by 25 mM K+. TPA (10 and 100 nM) did not significantly affect the basal [Ca2+]i in the afferent arteriole. The [Ca2+]i response to NA or 25 mM K+ was not affected by TPA. We conclude that blockade of PKC increases [Ca2+]i in afferent arteriolar smooth muscle by a mechanism independent of L-type voltage-sensitive calcium channels. Inhibition of PKC blunts the relative increase in [Ca2+]i in response to AII and, to a lesser extent, that induced by NA. We conclude that PKC might be important in modulating the calcium changes that occur in response to these vasoconstrictors.

Metabotropic synaptic regulation of intrinsic response properties of turtle spinal motoneurones.

1. The effect of a brief train of electric stimuli in the dorsolateral funiculus on the intrinsic response properties of turtle motoneurones was investigated in transverse sections of the spinal cord in vitro. 2. Even when glutamatergic, GABAergic and glycinergic ionotropic synaptic transmission was blocked by antagonists of AMPA, NMDA, glycine and GABA receptors, dorsolateral funiculus (DLF) stimulation induced a facilitation of plateau potentials during current clamp and the underlying inward current in voltage clamp. This facilitation lasted more than 10 s. 3. The plateau potential and the facilitation by DLF stimulation was absent in the presence of 10 microM nifedipine. The DLF-induced facilitation was reduced by antagonists of 5-HT1A, group 1 metabotropic glutamate receptors and muscarine receptors. 4. These findings suggest that the intrinsic properties of spinal motoneurones are dynamically regulated by afferent synaptic activity. These afferents can be of spinal and extraspinal origin. Continuous regulation of intrinsic response properties could be a mechanism for motor flexibility.

Tyrosine kinases modulate the activity of L-type calcium channels in vascular smooth muscle cells from rat portal vein

In a previous study, we demonstrated that extracellular application of the tyrosine kinase inhibitor genistein produced a dose-dependent inhibition of the macroscopic slow (L-type) Ca2+ currents of vascular smooth muscle (VSM) cells, and that daidzein, an inactive analog of genistein, had no such effect. These results suggested that the L-type Ca2+ channels in VSM cells may be modulated by endogenous tyrosine kinase activity. To confirm and extend those findings, the effect of genistein on the activity of single Ca2+ channels was examined in freshly isolated single VSM cells from rat portal vein, using the cell-attached patch-clamp technique. The pipette solution contained 90 mM Ba2+ as charge carrier and 0.5 microM Bay K 8644 (to enhance basal activity of the channels), and the bath contained 140 mM KCl to "zero" the resting membrane potential. Depolarizing pulses to 0 mV, from a holding potential of -80 mV, elicited inward unitary currents that were blocked by 1 microM nifedipine (n = 6). The slope conductance of the unitary Ca2+ currents gave a value of 21.5 +/- 0.4 pS (n = 9) for the Ca2+ channels. Bath application of genistein (50 microM) did not change the unit amplitude and slope conductance: the conductance in the presence of genistein was 22.2 +/- 0.5 pS (n = 6). However, compared with controls, the activity of single Ca2+ channels was significantly inhibited by genistein in a dose-dependent fashion. The ensemble-averaged currents were decreased by 48.4 +/- 11.2% with 50 microM genistein; 100 microM genistein inhibited the Ca2+ currents by 76.8 +/- 11.8%. The open probability (NPo) was decreased by 50 microM genistein from 0.24 +/- 0.09 to 0.11 +/- 0.07. Single-channel kinetic analysis showed that genistein decreased the mean open time and prolonged the mean closed time. The inhibitory effect of genistein on the Ca2+ channel activity occurred within 3 min, and it could be reversed by washout within 3-5 min. Daidzein, in concentrations up to 300 microM, produced no change in the activity of the single Ca2+ channels. These results demonstrate that genistein inhibits the activity of the L-type Ca2+ channels in VSM cells, suggesting that the availability of the channels for voltage activation may be maintained through tonic tyrosine kinase phosphorylation.

Relaxation of evoked contractile activity of isolated guinea-pig ileum by (+/-)-kavain.

Kava pyrones are the pharmacologically active compounds of Piper methysticum Forst. In the present study, the effect of the synthetic kava pyrone (+/-)-kavain was investigated on evoked contractile activity of isolated guinea-pig ileum. (+/-)-Kavain (1 microM-1 mM) dose-dependently reduced contractions of ileum evoked by carbachol (10 microM), by BAY K 8644 (0.3 microM), or by substance P (0.05 microM). (+/-)-Kavain also inhibited the contractile responses induced by raising the extracellular K+ concentration from 4 to 20 mM and by blocking the K+ channel by barium chloride (1 mM) or 4-aminopyridine (0.3 mM). After pre-incubation with 1 microM nifedipine, carbachol (1 microM) evoked 18.2 +/- 14.3% of contraction at control (i.e. prior pre-incubation with nifedipine). This remaining response was completely abolished by high concentrations of (+/-)-kavain (400 microM). After treatment of the longitudinal ileum strips with pertussis toxin (PTX), carbachol (1 microM) evoked 27.0 +/- 6.2% of the control response in untreated ileum. These contractions were also blocked by (+/-)-kavain (400 microM). However, (+/-)-kavain had no effect on the caffeine-induced (20 mM) contractions of ileum strips, which were permeabilized with digitonin or beta-escin. Moreover, it failed to affect Ca(2+)-evoked contractions of skinned muscles. These results suggest that the kava pyrone (+/-)-kavain may act in a non-specific musculotropic way on the smooth muscle membrane.

Morphological heterogeneity of renal glomerular arterioles and distinct [Ca2+]i responses to ANG II.

The present study compares cytosolic calcium concentration ([Ca2+]i) responses to angiotensin II (ANG II) of afferent (AA) and efferent arterioles (EA) by taking account of the localization and morphological differences of EA. In outer cortex, 1 nM ANG II induced smaller [Ca2+]i increases in thin EA than in AA[48 +/- 10 (n = 12) vs. 94 +/- 7 nM (n = 11); P < 0.001]. In inner cortex, two types of EA were considered, i.e., thin and muscular ones. The response to 1 nM ANG II was 35% lower in thin than in muscular EA (P < 0.05) but did not differ from that obtained with corresponding AA. In EA of the outer cortex, 1 microM nifedipine, a dihydropyridine blocker of voltage-operated channels (VOCC), did not affect calcium influx, which was suppressed by 1 mM NiCl2, a nonselective calcium entry blocker. In other arterioles, nifedipine inhibited by approximately 40% calcium entry, and remaining influx was blocked by NiCl2. These results indicate a relationship between the magnitude of [Ca2+]i responses, activation of dihydropyridine-sensitive VOCC by ANG II, and the muscular morphology in renal glomerular arterioles. They suggest that ANG II regulates differently local renal microcirculation. They do not, however, support the hypothesis of a greater sensitivity to ANG II of EA compared with the AA of a given nephron.

Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes.

The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea-pig ventricular myocytes was tested using whole-cell patch-clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L-type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch-activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)-Ca2+ exchanger. When Gd3+ is used as a blocker of stretch-activated channels its actions upon both Ica.L and IK must therefore be accounted for.

Regional differences in the role of the Ca2+ and Na+ currents in pacemaker activity in the sinoatrial node.

The effect of block of the L-type Ca2+ current by 2 microM nifedipine and of the Na+ current by 20 microM tetrodotoxin on the center (normally the leading pacemaker site) and periphery (latent pacemaker tissue) of the rabbit sinoatrial node was investigated. Spontaneous action potentials were recorded with microelectrodes from either an isolated right atrium containing the whole node or small balls of tissue (approximately 0.3-0.4 mm in diameter) from different regions of the node. Nifedipine abolished the action potential in the center, but not usually in the periphery, in both the intact sinoatrial node and the small balls. Tetrodotoxin had no effect, on electrical activity in small balls from the center, but it decreased the takeoff potential and upstroke velocity and slowed the spontaneous activity (by 49 +/- 10%; n = 11) in small balls from the periphery. It is concluded that whereas the L-type Ca2- current plays an obligatory role in pacemaking in the center, the Na+ current plays a major role in pacemaking in the periphery.

Existence of voltage-dependent Ca2+ channels in vestibular dark cells: cytochemical and whole-cell patch-clamp studies.

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.

In vitro adrenaline and collagen-induced mobilization of platelet calcium and its inhibition by naftopidil, doxazosin and nifedipine.

The aim of the study was to obtain further information regarding the modes of action of doxazosin, naftopidil and nifedipine on platelet function. We conducted an in vitro study of drug influences on adrenaline and collagen-induced mobilization of platelet calcium. In the presence of fibrinogen (300 micrograms ml-1) both collagen (5 micrograms ml-1) and adrenaline (16 microM) stimulated the aggregation of washed platelets. Collagen induced a transient rise (+4.97 +/- 0.63 microM) in platelet Ca2+ concentration, [Ca2+]i, as measured using the photoprotein aequorin, which coincided with the onset of aggregation. Adrenaline induced a smaller rise (+3.6 +/- 0.96 microM) which, however, occurred after the onset of aggregation. Naftopidil, an alpha 1-adrenoreceptor antagonist produced a concentration-dependent inhibition of collagen-induced Ca2+ mobilization, maximum inhibition (22.9 +/- 4%, P < 0.05) occurring with 40 microM naftopidil. The inhibition of Ca2+ mobilization was not reflected by a concentration-dependent inhibition of platelet aggregation, although 40 microM naftopidil produced statistically significant inhibition (23.3 +/- 11.7%, P < 0.05). The adrenaline-induced rise in [Ca2+]i was inhibited dose dependently by naftopidil (e.g. 40 microM naftopidil, 100 +/- 0%, P < 0.05), as was aggregation (40 microM naftopidil, 100 +/- 0%, P < 0.05). Doxazosin, another alpha 1-adrenoreceptor blocker, inhibited Ca2+ mobilization induced by collagen to similar extents as for naftopidil (30 microM doxazosin, 17.4 +/- 2.5%, P < 0.05), but did not inhibit platelet aggregation. It also inhibited the adrenaline-induced rise in [Ca2+]i in a concentration-dependent manner (30 microM doxazosin, 37.6 +/- 13.7%, P < 0.05), significant inhibitions of platelet aggregation also being produced (30 microM, 49.6 +/- 17.2%, P < 0.05). As expected, the calcium channel blocker nifedipine produced concentration-dependent inhibitions of both collagen-induced Ca2+ mobilization (e.g. 28 microM nifedipine, 47.8 +/- 2.7%, P < 0.05) and aggregation (28 microM, 55.1 +/- 9.2%, P < 0.05). These data indicate that the alpha 1-adrenoreceptor blockers, naftopidil and doxazosin, inhibit Ca2+ mobilization, this mechanism being possibly the means whereby these drugs inhibit platelet aggregation.

Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release. 2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 microM indomethacin, 300 microM NG-nitro-L-arginine or 10 microM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF. 3. In the presence of extracellular Ca2+, 1 microM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca(2+)-free medium, ACh produced only transient hyperpolarization. 4. Pretreatment with 100 nM thapsigargin and 3 microM cyclopiazonic acid, endoplasmic reticulum Ca(2+)-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 microM ryanodine. 5. In the presence of 5 mM Ni2+ or 3 mM Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 microM nifedipine had no effect on ACh-induced hyperpolarization. 6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 microM 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization. 7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni(2+)- and Mn(2+)-sensitive pathway distinct from L-type Ca2+ channels. The Ca(2+)-influx mechanism seems to be activated following IP3-induced depletion of the pool.

Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat.

1. The effects of niflumic acid, an inhibitor of calcium-activated chloride currents, were compared with the actions of the calcium channel blocker nifedipine on noradrenaline- and 5-hydroxytryptamine (5-HT)-induced pressor responses of the rat perfused isolated mesenteric vascular bed. 2. Bolus injections of noradrenaline (1 and 10 nmol) increased the perfusion pressure in a dose-dependent manner. Nifedipine (1 microM) inhibited the increase in pressure produced by 1 nmol noradrenaline by 31 +/- 5%. Niflumic acid (10 and 30 microM) also inhibited the noradrenaline-induced increase in perfusion pressure and 30 microM niflumic acid reduced the pressor response to 1 nmol noradrenaline by 34 +/- 6%. 3. The increases in perfusion elicited by 5-HT (0.3 and 3 nmol) were reduced by niflumic acid (10 and 30 microM) in a concentration-dependent manner and 30 microM niflumic acid inhibited responses to 0.3 and 3 nmol 5-HT by, respectively, 49 +/- 8% and 50 +/- 7%. Nifedipine (1 microM) decreased the pressor response to 3 nmol 5-HT by 44 +/- 9%. 4. In the presence of a combination of 30 microM niflumic acid and 1 microM nifedipine the inhibition of the pressor effects of noradrenaline (10 nmol) and 5-HT (3 nmol) was not significantly greater than with niflumic acid (30 microM) alone. Thus the effects of niflumic acid and nifedipine were not additive. 5. In Ca-free conditions the transient contractions induced by 5-HT (3 nmol) were not reduced by 30 microM niflumic acid, suggesting that this agent does not inhibit calcium release from the intracellular store or the binding of 5-HT to its receptor. 6. Niflumic acid 30 microM did not inhibit the pressor responses induced by KCl (20 and 60 mumol) which were markedly reduced by 1 microM nifedipine. In addition, 1 microM levcromakalim decreased pressor responses produced by 20 mumol KCl. These data suggest that niflumic acid does not block directly calcium channels or activate potassium channels. 7. It is concluded that niflumic acid selectively reduces a component of noradrenaline- and 5-HT-induced pressor responses by inhibiting a mechanism which leads to the opening of voltage-gated calcium channels. Our data suggest that the Ca(2+)-activated chloride conductance may play a pivotal role in the activation of voltage-gated calcium channels in agonist-induced constriction of resistance blood vessels.

Tyrosine kinases modulate the activity of L-type calcium channels in vascular smooth muscle cells from rat portal vein

In a previous study, we demonstrated that extracellular application of the tyrosine kinase inhibitor genistein produced a dose-dependent inhibition of the macroscopic slow (L-type) Ca2+ currents of vascular smooth muscle (VSM) cells, and that daidzein, an inactive analog of genistein, had no such effect. These results suggested that the L-type Ca2+ channels in VSM cells may be modulated by endogenous tyrosine kinase activity. To confirm and extend those findings, the effect of genistein on the activity of single Ca2+ channels was examined in freshly isolated single VSM cells from rat portal vein, using the cell-attached patch-clamp technique. The pipette solution contained 90 mM Ba2+ as charge carrier and 0.5 microM Bay K 8644 (to enhance basal activity of the channels), and the bath contained 140 mM KCl to "zero" the resting membrane potential. Depolarizing pulses to 0 mV, from a holding potential of -80 mV, elicited inward unitary currents that were blocked by 1 microM nifedipine (n = 6). The slope conductance of the unitary Ca2+ currents gave a value of 21.5 +/- 0.4 pS (n = 9) for the Ca2+ channels. Bath application of genistein (50 microM) did not change the unit amplitude and slope conductance: the conductance in the presence of genistein was 22.2 +/- 0.5 pS (n = 6). However, compared with controls, the activity of single Ca2+ channels was significantly inhibited by genistein in a dose-dependent fashion. The ensemble-averaged currents were decreased by 48.4 +/- 11.2% with 50 microM genistein; 100 microM genistein inhibited the Ca2+ currents by 76.8 +/- 11.8%. The open probability (NPo) was decreased by 50 microM genistein from 0.24 +/- 0.09 to 0.11 +/- 0.07. Single-channel kinetic analysis showed that genistein decreased the mean open time and prolonged the mean closed time. The inhibitory effect of genistein on the Ca2+ channel activity occurred within 3 min, and it could be reversed by washout within 3-5 min. Daidzein, in concentrations up to 300 microM, produced no change in the activity of the single Ca2+ channels. These results demonstrate that genistein inhibits the activity of the L-type Ca2+ channels in VSM cells, suggesting that the availability of the channels for voltage activation may be maintained through tonic tyrosine kinase phosphorylation.

L-type calcium current activation in cultured human myotubes.

The time course of activation of the skeletal muscle L-type calcium channel was studied in voltage-clamped myotubes derived from human satellite cells. The slow L-type current was isolated by inactivating faster calcium current components using appropriate prepulses or by subtracting the currents not blocked by 5 microM nifedipine. The L-type current exhibited a single exponential activation and time constants which showed little voltage dependence in the range +10 to +50mV. Currents blocked by nifedipine could be partially restored by UV-light flash photolysis. When a flash of light was applied during a depolarizing step, the activation time course of the resulting inward current contained a rapid, almost instantaneous component followed by a slower component. The amplitude of the rapid component was different when the flash was applied at different times during the depolarizing step: depolarization first increased and then decreased the fraction of channels which could rapidly be restored from the block by photolysis. Plotted versus time after the onset of the depolarization this fraction closely matched the time course of the L-type current obtained before the block by nifedipine. This indicates that the slow gating recations of the Ca2+ channel remain functional in the nifedipine-blocked state. Large conditioning depolarizations which had been shown to enhance the speed of L-type current activation in frog muscle fibres showed no effect in human myotubes. Numerical simulations using a gating scheme proposed for frog muscle demonstrate that such differences can be caused by changing just a single kinetic parameter.

Localization of voltage-sensitive Ca2+ fluxes and neuropeptide Y immunoreactivity to varicosities in SH-SY5Y human neuroblastoma cells differentiated by treatment with the protein kinase inhibitor staurosporine.

The distribution of voltage-sensitive elevations of the level of Ca2+ in untreated SH-SY5Y cells and cells that had been induced to differentiate with staurosporine was investigated by monitoring fura-2 fluorescence in cell suspensions, and by using microfluorometry and quantitative fluorescence imaging on cell bodies and on cellular processes. Cell bodies of both types of cells displayed small Ca2+ elevations, which were composed of transient and sustained components. Elevations were partially sensitive to the L- and N-channel blockers nifedipine (1 microM) and omega-conotoxin GVIA (100 nM) respectively. Up to ten times Ca2+ elevations were observed in varicosities of treated cells than in cell bodies of treated and cells. These elevations were insensitive to compounds known to release Ca2+ from intracellular stores. Elevations of Ca2+ were sustained, and they were insensitive to 5 microM nifedipine, 100 nM omega-agatoxin IVA and 100 nM omega-conotoxin GVIA, and partially sensitive to 2 microM omega-conotoxin GVIA, indicating predominance of non-L-type, non-N-type, non-P-type channel activity. The intracellular localization of neuropeptide Y, a marker of differentiation in these cells, was also investigated by fluorescence immunocytochemistry. Varicosities of treated cells displayed marked fluorescence when viewed in a confocal microscope. These findings show that the varicosities of staurosporine-treated cells exhibit some of the functional properties of nerve terminals. The varicosities resemble boutons en passant nerve endings and they seem to express Ca2+ channels different from those in the cell body.

BAY K 8644 and ClO4- potentiate caffeine contracture without Ca2+ release channel activation.

Effects of perchlorate (ClO4-) and BAY K 8644 on caffeine contracture and Ca2+ release channel current were studied in frog skeletal muscle. Single fibers produced a small transient contracture on addition of 2.2 mM caffeine. ClO4 at 10 mM enhanced caffeine contracture 3.7-fold. This effect was inhibited by 10 microM nifedipine pretreatment. An increase in caffeine contracture was also obtained after exposure to 0.1 microM BAY K 8644 for 1 h. At 20 mM, external K+ potentiated caffeine contracture 2.2-fold. ClO4- (< 10 mM) and BAY K 8644 (0.1-1 microM) did not affect open probability (Po), unitary conductance, and open and closed time constants of the Ca2+ release channel current. BAY K 8644 at 0.1 microM did not further enhance the channel that had been activated by 2 mM caffeine. However, 20-30 mM ClO4 increased Po significantly and led the channel to a long open state by increasing the slow open time constant and decreasing the fast closed time constant. These results suggest that binding of ClO4 and BAY K 8644 to dihydropyridine receptors elicits a further increase in Ca2+ release from the sarcoplasmic reticulum.

Hyperpolarization-activated Ca2+-permeable channels in the plasma membrane of tomato cells.

The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca2+ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel recordings, clamped membrane voltages more negative than -120 mV resulted in time- and voltage-dependent current activation. Single-channel currents saturated with increasing activities of Ca2+ and Ba2+ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca2+ or Ba2+, respectively. These channels were 20-25 and 10-13 times more permeable to Ca2+ than to K+ and to Cl-, respectively. Channel currents were strongly inhibited by 10 microM lanthanum and 50% inhibited by 100 microM nifedipine. This evidence suggests that hyperpolarization-activated Ca2+-permeable channels provide a mechanism for the influx of Ca2+ into tomato cells.

Canine bronchial sustained contraction in Ca2+-free medium: role of intracellular Ca2+

We evaluated whether cartilage was a source of Ca2+ and the possible role of Ca2+ recycling in the sustained bronchial contraction (SBC) induced by carbachol (Cch) in Ca2+-free medium. Canine first-order bronchi were studied with cartilage and epithelium (+CAR + EPI) and without these structures individually (-CAR + EPI and +CAR - EPI) or together (-CAR - EPI). After cartilage removal (-CAR - EPI or -CAR + EPI) Cch produced a transient contraction in Ca2+-free medium. Removal of the epithelium alone had minor effects on the magnitude of the SBC but increased the effect of removal of cartilage to diminish the SBC. Bronchial strips with cartilage were able to respond to Cch with lower Ca2+ concentrations (10-100 microM) than could dissected preparations. Preincubation with BAY K 8644 (30-1000 nM) or 60 mM KCl or -CAR - EPI tissues converted the transient contractions to Cch in Ca2+-free medium to sustained contractions. In microelectrode studies, 50 nM Cch induced membrane oscillations in solutions with 2.5 mM Ca2+ in bronchial preparations, plus or minus cartilage, and in undissected tissues in Ca2+-free medium but not in -CAR - EPI tissues. Preincubation with 1 microM BAY K 8644 in Ca(2+)-free medium restored these oscillations in -CAR - EPI tissues. The release of 45Ca2+ from cartilage was too rapid to provide a reservoir of Ca2+ to support multiple SBCs in Ca2+-free medium. Moreover, in the Ca2+-free medium (with 10 nM Ca2+ after tissue +CAR + EPI incubation) excitatory junction potentials rapidly disappeared. Addition of 1 microM nifedipine or 1 mM EGTA during the SBC of +CAR + EPI tissues produced complete relaxation. A transient contraction to Cch occurred with prior addition of nifedipine. Inhibition of the sarcoplasmic reticulum Ca2+ pump by tissue incubation with cyclopiazonic acid (CPA; 10 microM), or briefly with 1 mM EGTA significantly diminished the SBC induced by Cch in Ca2+-free medium. CPA and EGTA together abolished the Cch-induced SBC. Thus, cartilage plays a more complex role than as a Ca2+ reservoir to support the SBC induced by Cch in Ca2+-free solution; its removal affects the process supporting SBCs involving intracellular Ca2+ storage and Ca2+ entrance through voltage-dependent channels.