Opening Of Ca Channels Research Articles

Molecular mechanisms for the regulation of penile smooth muscle contractility

Relaxation of smooth muscle is viewed as a 'resetting' of contractile machinery and the resumption of a pre-contractile state is accomplished by lowering cytosolic Ca(2+) and/or by decreasing the sensitivity of the contractile machinery to Ca(2+). There are several mechanisms whereby cytosolic Ca(2+) can be reduced and relaxation achieved but, in general, all pathways depend upon the accumulation of cyclic nucleotides cAMP and cGMP or on the activation of K(+) channels resulting in hyperpolarization. Recently, activation of Na(+)/K(+) ATPase by nitric oxide has been shown to be involved in the relaxation of trabecular smooth muscle. Since Na(+)/K(+) ATPase is electrogenic, its stimulation would cause hyperpolarization and, in turn, would prevent the opening of voltage-dependent Ca(2+) channels. This manuscript briefly reviews the molecular mechanisms affected by muscle relaxants and vasodilators in the treatment of erectile dysfunction. International Journal of Impotence Research (2000) 12, Suppl 4, S34-S38.

Protein kinase calpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca(2+)](i).

Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Selective PKC isoform targeting may be a mechanism to achieve specificity. We used a green fluorescent fusion protein (GFP) to test the hypothesis that local changes in [Ca(2+)](i) regulate translocation of PKCalpha and that different modes of Ca(2+) and Ca(2+) release play a role in PKCalpha targeting. We constructed deletion mutants of PKCalpha to analyze the Ca(2+)-sensitive domains and their role in targeting. Confocal microscopy was used and [Ca(2+)](i) was measured by fluo-3. The fusion protein PKCalpha-GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wild-type PKCalpha protein. The Ca(2+) ionophore ionomycin induced a speckled cytosolic PKCalpha-GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage-operated Ca(2+) channel opening led to a localized accumulation of PKCalpha-GFP near the plasma membrane. Opening Ca(2+) stores with InsP(3), thapsigargin, or ryanodine induced a specific PKCalpha-GFP targeting to distinct intracellular areas. The G-protein-coupled receptor agonist thrombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca(2+) influx and led to a linear PKCalpha-GFP membrane association. PKCalpha-GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessary for Ca(2+)-induced PKCalpha targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKCalpha can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca(2+)](i). The targeting mechanism is dependent on the C2 and ATP binding site of the enzyme. Localized [Ca(2+)](i) changes determine the spatial and temporal targeting of PKCalpha.

Protein kinase Cα targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca2+]i

Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Selective PKC isoform targeting may be a mechanism to achieve specificity. We used a green fluorescent fusion protein (GFP) to test the hypothesis that local changes in [Ca(2+)](i) regulate translocation of PKCalpha and that different modes of Ca(2+) and Ca(2+) release play a role in PKCalpha targeting. We constructed deletion mutants of PKCalpha to analyze the Ca(2+)-sensitive domains and their role in targeting. Confocal microscopy was used and [Ca(2+)](i) was measured by fluo-3. The fusion protein PKCalpha-GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wild-type PKCalpha protein. The Ca(2+) ionophore ionomycin induced a speckled cytosolic PKCalpha-GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage-operated Ca(2+) channel opening led to a localized accumulation of PKCalpha-GFP near the plasma membrane. Opening Ca(2+) stores with InsP(3), thapsigargin, or ryanodine induced a specific PKCalpha-GFP targeting to distinct intracellular areas. The G-protein-coupled receptor agonist thrombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca(2+) influx and led to a linear PKCalpha-GFP membrane association. PKCalpha-GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessary for Ca(2+)-induced PKCalpha targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKCalpha can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca(2+)](i). The targeting mechanism is dependent on the C2 and ATP binding site of the enzyme. Localized [Ca(2+)](i) changes determine the spatial and temporal targeting of PKCalpha.

Potassium channel openers prevent β-amyloid toxicity in bovine vascular endothelial cells

There is increasing evidence that the cerebrovasculture may be involved in the pathology of Alzheimer's disease. Here, we report that potassium channel openers (KCOs) inhibit dose and time dependent necrosis induced by β-amyloid (Aβ) in cultured vascular endothelial cells. Cell proliferation rate was assayed by a colorimetric method. Aβ cytotoxicity and inhibition by the K ATP channel opener diazoxide and the K Ca channel opener NS1619 was correlated with changes in nitric oxide (NO) production. The protective effects were partly blocked by potassium channel blockers. Toxicity of Aβ and KCO protection was verified by histological examination of endothelial cells with scanning electron microscopy. eNOS levels in endothelial cells were not changed by any of the treatments. The results suggest that disruption of K + channels function may be a critical step in Aβ-induced cytotoxicity in endothelial cells by alteration of NO release.

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release.

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.

EDHF-mediated relaxation in rat gastric small arteries: influence of ouabain/Ba2+ and relation to potassium ions.

In several blood vessels, endothelium-dependent vasorelaxation is in part mediated by an endothelium-derived hyperpolarizing factor (EDHF), the nature of which is as yet unknown. Experiments were performed to investigate whether the recently raised hypothesis that EDHF might be identified as the potassium ion, released by activation of endothelial K(Ca) channels and inducing relaxation by stimulation of Na+/K+-pump and the inward rectifier K+ conductance, might be valid for small rat gastric arteries. EDHF-induced relaxation (assessed as the nitro-L-arginine/indomethacin resistant component of acetylcholine-induced relaxation), but not nitroprus-side-induced relaxation is strongly inhibited in the presence of ouabain (0.5 mM)/Ba2+ (30 microM), ouabain being responsible for the greater part of the inhibition. This inhibition is reversible. Application of increasing concentrations of K+ elicits transient relaxations in some preparations, but in a greater part of the preparations, no or only small relaxations. In membrane potential measurements, it was found that increasing concentrations of extracellular K+ consistently depolarized smooth muscle cells, whereas acetylcholine elicits hyperpolarization. The K(Ca) channel openers NS 1619 and 1-EBIO elicit relaxation effects that are not diminished after removal of the endothelium and are not inhibited by ouabain/Ba2+. It is concluded that EDHF-mediated relaxation is sensitive to inhibition by ouabain/Ba2+, but that the relation of this inhibitory influence to an action of K+ as EDHF is uncertain.

Protein-protein interactions in neurotransmitter release.

The arrival of a nerve impulse at a nerve terminal leads to the opening of voltage-gated Ca(2+) channels and a rapid influx of Ca(2+). The increase in Ca(2+) concentration at the active zone from the basal level of 100-200 mM triggers the fusion of docked synaptic vesicles, resulting in neurotransmitter release. A large number of proteins have been identified at nerve terminals and a cascade of protein-protein interactions has been suggested to be involved in the cycling of synaptic vesicle states. Functional studies in last half decade on synaptic-terminal proteins, including Ca(2+) channels, have revealed that the SNARE core complex, consisting of synaptobrevin VAMP, a synaptic vesicle-associated protein, syntaxin and SNAP-25, synaptic membrane-associated proteins, acts as the membrane fusion machinery and that proteins interacting with the SNARE complex play essential roles in synaptic vesicle exocytosis by regulating assembly and disassembly of the SNARE complex.

Dihydropyridine-sensitive Ca(2+) channels in human glomerular mesangial cells.

In mesangial cells (MC), the response of intracellular Ca(2+) concentration ([Ca(2+)](i)) to a contractile agonist is biphasic with a large, transient increase in [Ca(2+)](i) followed by a smaller but sustained elevation as Ca(2+) flows into the cell from the extracellular fluid. It has been postulated that membrane depolarization precedes opening of Ca(2+) channels in the plasmalemmal membrane. However, a role for voltage-gated Ca(2+) channels (VGCC) in human MC has been controversial, and their existence has not been verified with single-channel analysis. We used fura 2 fluorescence and patch-clamp techniques to determine the properties of the Ca(2+) entry pathway responsible for the sustained response of [Ca(2+)](i) in human MC. We found that ANG II at 10 nM, 100 nM, and 1 microM increased [Ca(2+)](i) to sustained levels of 22%, 35%, and 49%, respectively, above baseline. The sustained response to 1 microM ANG II was attenuated by diltiazem and was reduced to a value less than baseline in the absence of external Ca(2+). None of the peak responses (due to release of intracellular stores of Ca(2+)) were affected by removal of external Ca(2+) or addition of diltiazem. Upon elevating the extracellular [K(+)] from 5 mM to 75 mM, [Ca(2+)](i) reached a sustained level of 48% greater than baseline. This effect of high K(+) was attenuated by either Ca(2+) removal or addition of diltiazem. In the presence of 75 or 140 mM K(+), the dihydropyridine agonist BAY K 8644 (1 microM and 10 microM) initiated sustained [Ca(2+)](i) responses averaging 18% and 25%, respectively, greater than baseline. With <10 nM Ca(2+) in the external solution, BAY K 8644 did not significantly affect [Ca(2+)](i). In separate patch-clamp experiments, barium-selective channels were found in cell-attached patches with 90 mM BaCl(2) and 10 microM BAY K 8644 in the pipette solution. The single-channel conductance was 11.2 pS, and the open probability increased steeply at membrane potentials between -30 mV and 0 mV. It is concluded that human glomerular MC contain dihydropyridine-sensitive Ca(2+) channels responsible for the voltage-regulated entry of Ca(2+) into the cell during an agonist-induced contraction.

Influence of age on contractile response to insulin-like growth factor 1 in ventricular myocytes from spontaneously hypertensive rats.

Evidence suggests a pathophysiological role of insulin-like growth factor 1 (IGF-1) in hypertension. Cardiac function is altered with advanced age, similar to hypertension. Accordingly, the effects of IGF-1 on cardiac myocyte shortening and intracellular Ca(2+) were evaluated in hypertension at different ages. Ventricular myocytes were isolated from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), aged 12 and 36 weeks. Mechanical and intracellular Ca(2+) properties were examined by edge-detection and fluorescence microscopy. At 12 weeks, IGF-1 (1 to 500 ng/mL) increased peak twitch amplitude (PTA) and FFI changes (DeltaFFI) in a dose-dependent manner in WKY myocytes, with maximal increases of 27.5% and 35.2%, respectively. However, IGF-1 failed to exert any action on PTA and DeltaFFI in the age-matched SHR myocytes. Interestingly, at 36 weeks, IGF-1 failed to exert any response in WKY myocytes but depressed both PTA and DeltaFFI in a dose-dependent manner in SHR myocytes, with maximal inhibitions of 40.5% and 16.1%, respectively. Myocytes from SHR or 36-week WKY were less sensitive to norepinephrine (1 micromol/L) and KCl (30 mmol/L). Pretreatment with nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/L) did not alter the IGF-1-induced response in 12-week WKY myocytes but unmasked a positive action in 12-week SHR and 36-week WKY myocytes. L-NAME also significantly attenuated IGF-1-induced depression in 36-week SHR myocytes. In addition, the Ca(2+) channel opener Bay K8644 (1 micromol/L) abolished IGF-1-induced cardiac depression in 36-week SHR myocytes. Collectively, these results suggest that the IGF-1-induced cardiac contractile response was reduced with advanced age as well as with hypertension. Alterations in nitric oxide and intracellular Ca(2+) modulation may underlie, in part, the resistance to IGF-1 in hypertension and advanced age.

Semaphorin 3A growth cone collapse requires a sequence homologous to tarantula hanatoxin.

Axonal guidance is key to the formation of neuronal circuitry. Semaphorin 3A (Sema 3A; previously known as semaphorin III, semaphorin D, and collapsin-1), a secreted subtype of the semaphorin family, is an important axonal guidance molecule in vitro and in vivo. The molecular mechanisms of the repellent activity of semaphorins are, however, poorly understood. We have now found that the secreted semaphorins contain a short sequence of high homology to hanatoxin, a tarantula K(+) and Ca(2+) ion channel blocker. Point mutations in the hanatoxin-like sequence of Sema 3A reduce its capacity to repel embryonic dorsal root ganglion axons. Sema 3A growth cone collapse activity is inhibited by hanatoxin, general Ca(2+) channel blockers, a reduction in extracellular or intracellular Ca(2+), and a calmodulin inhibitor, but not by K(+) channel blockers. Our data support an important role for Ca(2+) in mediating the Sema 3A response and suggest that Sema 3A may produce its effects by causing the opening of Ca(2+) channels.

Release of Ca2+ from Intracellular Stores and Entry of Extracellular Ca2+ Are Involved in Sea Squirt Sperm Activation

A rise in intracellular free Ca2+ concentration ([Ca2+]i) is required to activate sperm of all organisms studied. Such elevation of [Ca2+]i can occur either by influx of extracellular Ca2+ or by release of Ca2+ from intracellular stores. We have examined these sources of Ca2+ in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca2+-sensitive dye fura-2 to monitor [Ca2+]i by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca2+], as well as by the presence of high [K+]o or the Ca channel blockers pimozide, penfluridol, or Ni2+, but not nifedipine or Co2+. The accompanying rise in [Ca2+]i was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca2+ entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca2+]i increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca2+]i could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca2+ from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP3) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca2+ and that thimerosal stimulates release of Ca2+ from IP3-sensitive intracellular stores.

Imaging Ca(2+) entering the cytoplasm through a single opening of a plasma membrane cation channel.

Discrete localized fluorescence transients due to openings of a single plasma membrane Ca(2+) permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca(2+) indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375-394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca(2+), suggesting that they are due to Ca(2+) influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca(2+) and Ca(2+)-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca(2+) and Ca(2+)-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a "Ca(2+) spark-like" appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca(2+) current, to study intracellular localized Ca(2+) handling as might occur from single Ca(2+) channel openings, and to localize Ca(2+) permeable ion channels on the plasma membrane.

Mechanisms of hypoxic coronary vasodilatation in isolated perfused rat hearts.

We pharmacologically investigated the potential involvement of nitric oxide (NO), prostacyclin, adenosine, adenosine triphosphate (ATP)-sensitive K (K(ATP)) channel opening and Ca2+-activated K (K(Ca)) channel opening in coronary vasodilatation during 15 min of hypoxia in isolated rat hearts perfused at a constant pressure of 70 mm Hg. The coronary flow suppressed by 10(-4) M Nomega-nitro-L-arginine methyl ester (L-NAME), which corresponds to the NO-dependent flow, decreased to almost zero during hypoxia. In contrast, the NO-dependent coronary flow amounted to approximately 40% of the total coronary flow during normoxia. The suppression of coronary flow by 10(-5) M 8-phenyltheophylline (8-PT), which corresponds to the adenosine-dependent flow, was remarkable in the middle and the late phases of a 15-min hypoxia. The coronary flow suppressed by 2 x 10(-6) M glibenclamide, which corresponds to the K(ATP) channel opening-dependent flow, depended on the agents added to the perfusate. However, there was a marked increase in coronary flow in the early phase of hypoxia in the heart perfused with the combination of 8-PT, 10(-2) M tetraethylammonium (TEA) and L-NAME. During hypoxia, the coronary flow suppressed by TEA, which corresponds mainly to the K(Ca) channel opening-dependent flow, also depended on the agents added to the perfusate. However, during reoxygenation, there was a transient significant increase in any combination of the agents. Our study suggests that hypoxia almost completely inhibits NO production, and that K(ATP) channel opening immediately after hypoxia and subsequent enhanced adenosine production cause a marked hypoxic coronary vasodilatation. It also suggests that K(Ca) channel opening causes vasodilatation during reoxygenation.

Calcium signaling and cytotoxicity.

The divalent calcium cation Ca(2+) is used as a major signaling molecule during cell signal transduction to regulate energy output, cellular metabolism, and phenotype. The basis to the signaling role of Ca(2+) is an intricate network of cellular channels and transporters that allow a low resting concentration of Ca(2+) in the cytosol of the cell ([Ca(2+)]i) but that are also coupled to major dynamic and rapidly exchanging stores. This enables extracellular signals from hormones and growth factors to be transduced as [Ca(2+)]i spikes that are amplitude and frequency encoded. There is considerable evidence that a number of toxic environmental chemicals target these Ca(2+) signaling processes, alter them, and induce cell death by apoptosis. Two major pathways for apoptosis will be considered. The first one involves Ca(2+)-mediated expression of ligands that bind to and activate death receptors such as CD95 (Fas, APO-1). In the second pathway, Ca(2+) has a direct toxic effect and its primary targets include the mitochondria and the endoplasmic reticulum (ER). Mitochondria may respond to an apoptotic Ca(2+) signal by the selective release of cytochrome c or through enhanced production of reactive oxygen species and opening of an inner mitochondrial membrane pore. Toxic agents such as the environmental pollutant tributyltin or the natural plant product thapsigargin, which deplete the ER Ca(2+) stores, will induce as a direct result of this effect the opening of plasma membrane Ca(2+) channels and an ER stress response. In contrast, under some conditions, Ca(2+) signals may be cytoprotective and antagonize the apoptotic machinery.

Effects of SCA40 on bovine trachealis muscle and on cyclic nucleotide phosphodiesterases

While UK-93,928 (1-[[3-(6,9-dihydro-6-oxo-9-propyl-1H-purin-2-yl)-4-ethoxyphenyl] sulfonyl]-4-methylpiperazine; 5 nM–5 μM) was devoid of relaxant activity, benzafentrine, isoprenaline, levcromakalim and SCA40 (6-bromo-8-methylaminoimidazo[1,2- a]pyrazine-2-carbonitrile) each relaxed histamine (460 μM)-precontracted bovine isolated trachealis. Each of these relaxants was antagonised by a K +-rich (80 mM) medium. Except in the case of levcromakalim, nifedipine (1 μM) offset this antagonism. Charybdotoxin (100 nM) antagonised isoprenaline in a nifedipine-sensitive manner but did not antagonise SCA40 or benzafentrine. Iberiotoxin (100 nM) did not antagonise SCA40. Acting on tissue precontracted with carbachol, SCA40 potentiated isoprenaline but did not potentiate sodium nitroprusside. While levcromakalim (1 and 10 μM) induced hyperpolarisation, SCA40 (1 and 10 μM) induced little change in the membrane potential of bovine trachealis. In trachealis preloaded with 86Rb + , levcromakalim (1 and 10 μM) promoted efflux of the radiotracer while SCA40 (1 and 10 μM) had no effect. Tested as an inhibitor of isoenzymes of cyclic nucleotide phosphodiesterase, SCA40 was most potent against the type III, less potent against the type IV and least potent against the type I isoenzyme. It is concluded that neither inhibition of phosphodiesterase type V nor the promotion of BK Ca channel opening explains the tracheal smooth muscle relaxant activity of SCA40. This compound relaxes bovine tracheal smooth muscle mainly by inhibiting phosphodiesterase isoenzyme types III and IV.

Serotonin induces inward potassium and calcium currents in rat cortical astrocytes

Ca 2+ imaging and patch-clamp techniques were used to study the effects of serotonin (5-HT) on ionic conductances in rat cortical astrocytes. 1 and 10 μM serotonin caused a transient increase in intracellular calcium (Ca 1) levels in fura-2AM-loaded cultured astrocytes and in astrocytes acutely isolated and then cultured in horse serum-containing medium for over 24 h. However, the acutely isolated (less than 6 h from isolation) astrocytes, as well as acutely isolated astrocytes cultured in serum-free media, failed to respond to 5-HT by changes in Ca 1. Coinciding with the changes in Ca 1 levels, inward currents were activated by 10 μM 5-HT in cultured, but not in acutely isolated astrocytes. Two separate types of serotonin-induced, small-conductance inward single-channel currents were found. First, in both Ca 2+-containing and Ca 2+-free media serotonin transiently activated a small-conductance apamin-sensitive channel. Apamin is a specific blocker of the small-conductance Ca 2+-activated K + channel (sK Ca). When cells were pre-treated with phospholipase C inhibitor U73122 no 5-HT-induced sK Ca channel openings were seen, indicating that this channel was activated by Ca 2+ released from intracellular stores via IP 3. A second type of small inward channel activated later, but only in the presence of external Ca 2+. It was inhibited by the L-type Ca 2+ channel blockers, nimodipine and nifedipine. Both types of channel activity were inhibited by ketanserin, indicating activation of the 5-HT 2A receptor.

Activation of large conductance potassium channels inhibits the afferent and efferent function of airway sensory nerves in the guinea pig.

Sensory nerves play an important role in airway disease by mediating central reflexes such as cough, and local axon reflexes resulting in the peripheral release of neuropeptides. We have tested whether the benzimidazolone compound, NS1619, an opener of large conductance calcium-activated potassium (BK Ca) channels, inhibits the activity of sensory fibers, and central and local airway reflexes in guinea pig airways. In in vitro single fiber recording experiments, NS1619 applied to identified receptive fields in the trachea inhibited the firing of A(delta)-fibers evoked by hypertonic saline and distilled water, and bradykinin-evoked firing of C-fibers. Electrically evoked nonadrenergic noncholinergic contractions of isolated bronchi mediated by the release of neurokinin A (NKA) from C-fibers, but not those elicited by exogenous NKA, were inhibited by NS1619. These effects of NS1619 were prevented by iberiotoxin, a selective blocker of BK Ca channels. In conscious guinea pigs, cough evoked by aerosolized citric acid was also inhibited by NS1619. These data show that BK Ca channel activation inhibits sensory nerve activity, resulting in a reduction of both afferent and efferent function. BK Ca channel openers may therefore be of potential benefit in reducing neurogenic inflammation and central reflexes seen during inflammatory conditions of the airways, and may represent a new class of antitussive drug.

On the mechanism of the differential effects of NS004 and NS1608 in smooth muscle cells from guinea pig bladder

Our recent study revealed that the reported BK Ca channel openers NS004 (5-trifluoromethyl-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2 H-benzimidazole-2-one) and its analog NS1608 ( N-(3-(trifluoromethyl)phenyl)- N′-(2-hydroxy-5-chlorophenyl)urea) produced a differential pattern of action on the BK Ca channel in porcine coronary arterial cells (Hu, S., H.S. Kim and C.A. Fink, 1995, Differential effects of the BK Ca channel openers NS004 and NS1608 in porcine coronary arterial cells, Eur. J. Pharmacol. 294, 357). In this study, using the patch-clamp method on the smooth muscle cells from guinea pig bladder detrusor, the activity profile of NS004 and NS1608 on the whole-cell BK Ca current ( I BK) was examined and found to be similar to that in coronary arterial cells. NS004 did not significantly modify I BK at concentrations below 5 μM, but robustly augmented I BK at concentrations greater than 20 μM. With a minimum effective concentration of 0.5 μM. NS1608 caused potentiation of I BK with a bell-shaped concentration-response relationship. The activation was maximized between 5–10 μM and substantially attenuated at higher concentrations. The behavior of single BK Ca channels in the presence of NS004 and NS1608 was scrutinized to elucidate the mechanism underlying their distinct patterns of action. The channel open-state probability (NP o) was increased by NS004 at 0.5, 5 and 50 μM to a respective 1.75 ± 0.38, 4.05 ± 0.90, and 15.01 ± 3.66 fold ( n = 7) of the control by increasing the open time and the frequency of opening while having no effect on the single BK Ca channel conductance. NS1608 at 0.5 and 5 μM increased NP o to 1.69 ± 0.43 and 18.03 ± 6.64 fold of the control ( n = 4), respectively. NS1608 at higher concentrations repeatedly blocked channel openings as evidenced by the highly flickering open state, though the overall open time and frequency of opening remained high. The diminished activation of I BK by 50 μM NS1608 manifested therefore a net result of these two opposing actions on the single channels. Thus, the two structurally related BK Ca channel openers interact distinctly with the channel gating components.

The in vitro action of polyamines on rat basilar and femoral artery contractile activity

This study was performed to assess the role of exogenously administered polyamines on rat basilar and femoral artery contractile activity in vitro. With the endothelium removed, rings of tissue were set up in organ chambers to measure isometric tension. The polyamines (0.1–3mM), putrescine, spermidine, and spermine, were added to the tissue baths; after 30 min of incubation a cumulative concentration response curve (CRC) was obtained with either KCl or serotonin (5-HT). Additional CRCs were run with Ca ++ in high K + Krebs (60mM). In both tissues, the CRCs to KCl were shifted to the right in a dose-dependent manner for spermidine and spermine (1 & 3 mM) but not putrescine. Spermine (3mM) depressed the KCl maxima by 18.6% and 10.1% in the basilar and femoral artery respectively. For 5-HT CRCs, only spermine (3mM) slightly inhibited the maximal response in both tissues. The most potent action of spermine was on inhibition of Ca ++ responses in high K +where the EC 50s were shifted 3.5 and 10 fold over control values in the basilar and femoral respectively. We conclude spermidine and spermine, but not putrescine, attenuate vascular smooth muscle contractions on the basilar and femoral arteries in vitro. The exact nature of the inhibition remains to be fully explored, but blockade of calcium entry through voltage operated Ca channels may play a role. Thus, certain polyamines may affect cerebral perfusion by inhibition of vascular contractility.

Effect of Leiurus quinquestriatus hebreus venom on calcium and deoxyglucose uptake in cultured cardiac cells

The effects of scorpion venom Leiurus quinquestriatus hebreus were studied on cardiac cells grown in culture. The venom (30 μg/ml) increased significantly ( P < 0.05) Ca 2+ uptake into intact cardiocytes and to sarcoplasmic reticulum of skinned cells. [ 3H]Deoxyglucose uptake was also increased significantly ( P < 0.05) in venom treated cardiocytes. It was found that fractions I and III of the venom, separated by gel filtration and ion exchange chromatography, are responsible for the increased Ca 2+ uptake by the sarcoplasmic reticulum, whereas fraction IIb, III and IV are responsible for the accelerated rate of uptake of 45Ca and [ 3H]deoxyglucose by intact cells. Ca channel blockers prevented these effects and similar results were obtained by propranolol. Thus, it is concluded that the venom exerts its effect through activation of β-adrenoceptors which causes the opening of L-type Ca channels.