Apamin-sensitive Ca2 Research Articles

Mechanisms underlying hyperpolarization evoked by P2Y receptor activation in mouse distal colon

In murine colonic circular muscle, ATP mediates fast component of the nerve-evoked inhibitory junction potentials, via activation of P2Y receptors and opening of apamin-sensitive Ca 2+-dependent K + channels. We investigated, using microelectrode recordings, the intracellular events following P2Y-receptor activation by electrical field stimulation or by adenosine 5′- O-2-thiodiphosphate (ADPβS), ATP stable analogue. The fast-inhibitory junction potential amplitude was reduced by thapsigargin or ciclopiazonic acid (CPA), sarcoplasmic reticulum Ca 2+-ATPase inhibitors, by ryanodine, which inhibits Ca 2+ release from ryanodine-sensitive stores, and by 9-(tetrahydro-2-furanyl)-9 H-purin-6-amine (SQ 22,536), an adenylyl cyclase inhibitor. Fast-inhibitory junction potentials were enhanced by 2-aminoethoxy-diphenylborate (2-APB), an IP 3 receptor inhibitor or by {1-[6((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1 H-pyrrole-2,5-dione} (U-73122), a phospholipase C inhibitor. ADPβS induced hyperpolarization that was significantly reduced by apamin, thapsigargin, CPA, ryanodine, 2-APB and SQ 22,536, but it was not modified by U-73122. Forskolin, an adenylyl cyclase activator, induced hyperpolarization that was inhibited by SQ 22,536, apamin or ryanodine. In conclusion, in murine colon, apamin-sensitive hyperpolarization induced by activation of P2Y receptors is mainly mediated by release of Ca 2+ from intracellular ryanodine-dependent stores via a mechanism involving adenylyl cyclase.

Mechanisms underlying the inhibitory effects induced by pituitary adenylate cyclase-activating peptide in mouse ileum

The aim of this study was to investigate the signal transduction mechanisms underlying the inhibitory effect induced by pituitary adenylate cyclase activating peptide (PACAP-27) on the spontaneous contractile activity of longitudinal muscle of mouse ileum. Mechanical activity of ileal segments was recorded isometrically in vitro. PACAP-27 produced apamin-sensitive reduction of the amplitude of the spontaneous contractions. 9-(Tetrahydro-2-furanyl)-9 H-purin-6-amine (SQ 22,536), adenylate cyclase inhibitor, or genistein and tyrphostin 25, tyrosine kinase inhibitors, had negligible effects on PACAP-27-induced inhibition. PACAP-27 effects were significantly inhibited by U-73122, phopholipase C (PLC) inhibitor, by 2-aminoethoxy-diphenylborate (2-APB), permeable blocker of inositol 1,4,5-triphosphate (IP 3) receptors and by depletion of Ca 2+ stores with cyclopiazonic acid or thapsigargin. Ryanodine did not reduce PACAP-27-inhibitory responses. We suggest that, in mouse ileum, the inhibitory responses to PACAP-27 involve stimulation of PLC, increased production of IP 3 and localised Ca 2+ release from intracellular stores, which could provide the opening of apamin-sensitive Ca 2+-dependent K + channels.

Spontaneous Activity and Properties of Two Types of Principal Neurons From the Ventral Tegmental Area of Rat

We investigated the spontaneous activity and properties of freshly isolated ventral tegmental area (VTA) principal neurons by whole cell recording and single-cell RT-PCR. The VTA principal neurons, which were tyrosine hydroxylase-positive and glutamic acid decarboxylase (GAD67)-negative, exhibited low firing frequency and a long action potential (AP) duration. The VTA principal neurons exhibited a calretinin-positive and parvalbumin-negative Ca2+-binding protein mRNA expression pattern. The VTA principal neurons were classified into two subpopulations based on their firing frequency coefficient of variation (CV) at room temperature (21-23 degrees C): irregular-type neurons with a large CV and tonic-type neurons with a small CV. These two firing patterns were also recorded at the temperature of 34 degrees C and in nystatin-perforated patch recording. In VTA principal neurons, the AP afterhyperpolarization (AHP) amplitude contributed to the firing regularity and AHP decay slope contributed to the firing frequency. The AHP amplitude in the irregular-type VTA principal neurons was smaller than that in the tonic-type VTA principal neurons. There was no significant difference in the AHP decay slope between the two-types of VTA principal neurons. Apamin-sensitive small-conductance Ca2+-activated K+ (SK) channels contributed to the AHP and the regular firing of the tonic-type neurons but contributed little to the AHP and firing of the irregular-type neurons. In voltage-clamp tail-current analysis, in both conventional and nystatin-perforated whole cell recording, the apamin-sensitive AHP current density of the tonic-type neurons was significantly larger than that of the irregular-type neurons. We suggest that apamin-sensitive SK current contributes to intrinsic firing differences between the two subpopulations of VTA principal neurons.

Evidence for Domain-specific Recognition of SK and Kv Channels by MTX and HsTx1 Scorpion Toxins

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.

Characterization of the outer pore region of the apamin-sensitive Ca 2+-activated K + channel rSK2

We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K + channels. Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2. In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K + channel SK1 and SK3 like. Directed by the results of the toxin receptor on the ShakerK + channel, we changed single amino acids of the SK2 K + channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3. Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK + channel to improve Shaker K + channel CTX sensitivity. Interestingly SK2 V342G became CTX sensitive with a K d of 19 nM and was also KTX sensitive K d =97 nM. SK2 S344E (K d CTX =105 nM,K d KTX =144 nM ) and G348D ( K d CTX =31 nM,K d KTX=89 nM ) became also CTX and KTX sensitive with a lower affinity. The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity ( K d =6 nM,K d =48 nM, and K d =12 nM ). Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K + channel pore is very similar to those of voltage-gated K + channels such as the Shaker K + channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel. From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore.

Characterization of the outer pore region of the apamin-sensitive Ca2+-activated K+ channel rSK2

Characterization of the outer pore region of the apamin-sensitive Ca2+-activated K+ channel rSK2

Mechanisms underlying endothelium-dependent flow increase in perfused rat mesenteric vascular bed

The isolated rat mesenteric vasculature was perfused at constant pressures of 40, 80 or 120 mm Hg and the change in flow rate was measured. In the presence of phenylephrine, treatment with 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) or N G-nitro- l-arginine ( l-NA) significantly inhibited the pressure-dependent flow rate increase, but treatment with indomethacin or charybdotoxin plus apamin did not. Acetylcholine, bradykinin and ADP increased the flow rate, which had been markedly suppressed by CHAPS. At 80 mm Hg, the flow rate increase induced by these agonists was not affected by indomethacin plus l-NA, but was suppressed by subsequent treatment with charybdotoxin plus apamin. Changes in the perfusion pressure did not significantly affect the flow rate increases induced by the agonists. In conclusion, the opening of charybdotoxin plus apamin-sensitive Ca 2+-dependent K + channels may be mainly involved in the endothelium-dependent flow rate increase induced by the agonists, whereas nitric oxide (NO) may be responsible for the endothelium-dependent, pressure-induced flow rate increase.

Interplay between PACAP and NO in mouse ileum

We investigated the possibility that pituitary adenylate cyclase activating peptide (PACAP) has a role in the control of contractility in the mouse ileum. PACAP-(1-27) produced tetrodotoxin (TTX)-insensitive, concentration-dependent reduction of the amplitude of the spontaneous contractions of longitudinal muscle up to their complete disappearance. This effect was inhibited by PACAP-(6-38), PACAP receptor antagonist, and by apamin, blocker of small-conductance Ca 2+-activated K +-channels. N ω-nitro- l-arginine methyl ester (L-NAME), nitric oxide (NO) synthase inhibitor, reduced the PACAP-inhibitory response, and the joint application of apamin plus L-NAME produced additive effects. 1H-[1,2,4] oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), inhibitor of NO-stimulated soluble guanylate cyclase, significantly reduced the effect of PACAP. Exogenous NO, given as sodium nitroprusside (SNP), induced a concentration-dependent suppression of the phasic contractions, which was unaffected by apamin but reduced by either PACAP-(6-38) or TTX. Neurally evoked muscular relaxation was deeply antagonised by L-NAME. PACAP-(6-38) induced a reduction of the response to EFS only in the absence L-NAME. In conclusion, our results suggest that PACAP controls smooth muscle contractility, acting directly on the muscle cells through PACAP-27 preferring receptors coupled to apamin-sensitive Ca 2+-dependent K +-channels and indirectly through the stimulation of NO production. In turn, NO would stimulate the release of PACAP from inhibitory neurones.

Role of the cholinergic system and of apamin-sensitive Ca2+-activated K+ channels on rabbit jejunum spontaneous activity and on the inhibitory effects of adrenoceptor agonists.

1. One reason why rabbit jejunum is suitable for studying the mechanisms underlying the actions of the various neurotransmitters and their interactions is its spontaneous motility. The main regulator of spontaneous motility is the cholinergic system. How the cholinergic system regulates the spontaneous activity in the rabbit jejunum and how it affects the inhibitory action of alpha- and beta-adrenoceptor agonists remains unclear. 2. We studied the influence of the cholinergic system and apamin-sensitive Ca2+-activated K+ channels on spontaneous contractions in the rabbit jejunum and on the inhibitory effects of alpha1- and beta-adrenoceptor agonists. 3. In naïve tissues, atropine (ATR, 7.4 x 10(-8) m) and tetrodotoxin (8 x 10(-8) m) almost completely inhibited - to a similar extent - the amplitude of spontaneous activity. Despite the presence of ATR or TDX, tissue contraction gradually recovered to about 50% of the baseline amplitude within 5-10 min. When ATR or TDX, respectively, were added to the TDX- or ATR-treated tissues, the recovered activity decreased weakly but significantly. After washout and a 45-min rest the contraction amplitude returned to baseline values. A further exposure to ATR or TDX reduced the contraction to a level significantly lower than the one obtained after TDX or ATR added 5 min after ATR or TDX, respectively. In preparations prestimulated for 10 min with acetylcholine (ACh), ATR abolished the TDX-resistant recovered spontaneous activity. 4. Adrenaline (ADR, 0.5-5 x 10(-7) m) and phenylephrine (PHE, 1-10 x 10(-7) m) inhibited tissue motility in naïve and in ATR- and in TDX-exposed preparations. But whereas in naïve preparations the alpha1-adrenoceptor antagonists completely antagonized inhibition induced by both drugs, in ATR- and TDX-exposed tissues they did so only partially for ADR. Agonist-induced inhibition had a rapid onset but rapidly faded; pendular movements took significantly longer to recover in ATR- and TDX-treated tissues than in naïve tissues. In tissues exposed for 2 min to ADR (0.5-5 x 10(-7) m) or PHE (1-10 x 10(-7) m), washout or addition of alpha1-adrenoceptor antagonists caused an immediate short-lasting increase in contraction amplitude. 5. Apamin (APAM, 5 x 10(-9) m) caused a rapid and persistent increase in the amplitude of contractions. It also blocked the inhibitory responses to ADR and PHE, and removed washout-induced contractions. The APAM-induced increase in the contraction amplitude correlated with the increase obtained by washing out ADR or PHE. 6. Isoprenaline (at concentrations up to 2.8 x 10(-7) m) produced no inhibitory response in naïve tissues, but it invariably blocked (at a concentration of 0.7 x 10(-7) m) the recovered spontaneous activity (and sometimes depressed muscletone) in tissues exposed to ATR or TDX. Neither propranolol (3.4 x 10(-7) m) nor APAM (5 x 10(-9) m) counteracted these inhibitory effects. 7. These results indicate that spontaneous motility in the rabbit jejunum is predominantly mediated by neuronal release of ACh and by some other unidentified neuronal activity. Released ACh inhibits myogenic activity and strongly antagonizes beta-adrenoceptor-induced APAM-insensitive inhibition but leaves alpha1 agonist-induced APAM-sensitive inhibition unchanged.

γ-hydroxybutyrate receptor function determined by stimulation of rubidium and calcium movements from NCB-20 neurons

γ-Hydroxybutyrate is derived from GABA in brain and plays specific functional roles in the CNS. It is thought to exert a tonic inhibitory control on dopamine and GABA release in certain brain areas, through specific γ-hydroxybutyrate receptors. Apart from modifying certain calcium currents, the specific transduction mechanism induced by stimulation of γ-hydroxybutyrate receptors remains largely unknown. We investigated the possible contribution of K + channels to the hyperpolarization phenomena generally induced by γ-hydroxybutyrate in brain, by monitoring 86Rb + movements in a neuronal cell line (NCB-20 cells), which expresses γ-hydroxybutyrate receptors. Physiological concentrations of γ-hydroxybutyrate (5–25 μM) induce a slow efflux of 86Rb +, which peaks at 5–15 min and returns to baseline levels 20 min later after constant stimulation. This effect can be reproduced by the γ-hydroxybutyrate receptor agonist NCS-356 and blocked by the γ-hydroxybutyrate receptor antagonist 6,7,8,9-tetrahydro-5-[ H]-benzocycloheptene-5-ol-4-ylidene. The GABA B receptor antagonist CGP 55,845 has no effect on γ-hydroxybutyrate-induced 86Rb + efflux. The pharmacology of this γ-hydroxybutyrate-dependent efflux of 86Rb + is in favor of the involvement of tetraethylammonium and charybdotoxin insensitive, apamin sensitive Ca 2+ activated K + channels, identifying them as small conductance calcium activated channels. We demonstrated a γ-hydroxybutyrate dose-dependent entry of calcium ions into NCB-20 neuroblastoma cells at resting potential. Electrophysiological data showed that this Ca 2+ entry corresponded mainly to a left-hand shift of the current/voltage relation of the T-type calcium channel. This process must at least partially trigger small conductance calcium activated channel activation leading to γ-hydroxybutyrate-induced hyperpolarization.

Electrophysiological properties of inhibitory junction potential in murine lower oesophageal sphincter.

The electrophysiological properties of smooth muscle in the murine lower oesophageal sphincter (LOS) were investigated by intracellular microelectrode recording. Inhibitory junction potentials (IJPs) evoked by trains of field stimulation (30 V, 0.2-0.3 ms, 10 stimuli at 1-50 Hz) were observed in the murine LOS in the presence of atropine (1 microM) and nifedipine (1 microM). The IJP consists of two components, which we termed fast IJP and slow IJP. The fast IJP was partly sensitive to guanethidine (5 microM), pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) and apamin (0.1 microM), suggesting that the fast IJP was produced partly through the activation of apamin-sensitive Ca2+-activated K+ channels and of P2-purinoceptors. The other part of the fast IJP was sensitive to N(omega)-nitro-L-arginine (L-NNA, 100 microM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 1 microM), but insensitive to apamin (0.1 microM), iberiotoxin (50 nM) and charybdotoxin (30 nM). Slow IJP was sensitive to L-NNA (100 microM), ODQ (10 microM) and glibenclamide (10 microM), but insensitive to apamin (0.1 microM), iberiotoxin (50 nM) and charybdotoxin (30 nM). KT5823, a protein kinase G (PKG) inhibitor, had no effect on the fast and slow IJP in this tissue. It was suggested that, in the mouse LOS, adenosine trisphosphate (ATP) partly mediated the fast UP through apamin-sensitive Ca2+-activated K+ channels, and nitric oxide mediated the remained part of the fast IJP and the slow IJP through cGMP, but not PKG. ATP-sensitive K+ channels were suggested to be partly involved in the production of slow IJP, but the responsible channel(s) for the nitrergic fast IJP remained unclarified.

Mechanisms of the modulation of pacemaker activity by GnRH peptides in the terminal nerve-GnRH neurons.

According to our working hypothesis, the terminal nerve (TN)-gonadotropin releasing hormone (GnRH) system functions as a neuromodulatory system that regulates many long-lasting changes in animal behaviors. We have already shown by using in vitro whole brain preparations of a small fish (dwarf gourami) that the pacemaker activities of TN-GnRH neurons are modulated biphasically by salmon GnRH, which is the same molecular species of GnRH produced by TN-GnRH neurons themselves; the modulation consists of initial transient decrease and late increase of firing frequency. In the present study, we investigated the possible involvement of Ca2+ release from intracellular store and voltage dependent Ca2+ currents in the modulation of pacemaker activities. Pharmacological blockade of Ca2+ release from intracellular stores or apamin-sensitive Ca(2+)-activated K+ current inhibited the initial transient decrease of firing frequency by sGnRH. On the other hand, bath application of Ca2+ channel blockers Ni2+ or La3+ slowed down the pacemaker frequency and attenuated the rate of the late increase of pacemaker frequency by GnRH. Furthermore, voltage-clamp experiments suggested that low-voltage-activated (LVA) Ca2+ current and hihg-voltage-activated (HVA) Ca2+ current were present in the TN-GnRH neurons, and bath application of GnRH shifted the activation threshold of HVA Ca2+ current to more negative potentials. These results suggest that (1) sGnRH induces Ca2+ release from intracellular stores and activates apaminsensitive Ca(2+)-activated K+ current so that it decreases the frequency of pacemaker activity in the initial phase, (2) some kinds of Ca2+ currents contribute to the generation and modulation of pacemaker activities, and (3) HVA Ca2+ current is facilitated by sGnRH so that it increases the frequency of pacemaker activity in the late phase.

Differential modulation of respiratory neuronal discharge patterns by GABA(A) receptor and apamin-sensitive K(+) channel antagonism.

The discharge patterns of respiratory neurons of the caudal ventral respiratory group (cVRG) appear to be subject to potent GABAergic gain modulation. Local application of the GABA(A) receptor antagonist bicuculline methochloride amplifies the underlying discharge frequency (F(n)) patterns mediated by endogenous excitatory and inhibitory synaptic inputs. Gain modulation can also be produced by alterations in the amplitude of spike afterhyperpolarizations (AHPs) mediated by apamin-sensitive small-conductance Ca(2+)-activated K(+) (SK) channels. Since methyl derivatives of bicuculline (BICm) also have been shown to reduce the amplitude of AHPs, in vitro, it is possible that the BICm-induced gain modulation is due to a block of SK channels. The purpose of these studies was to determine the mechanisms by which BICm produces gain modulation and to characterize the influence of SK channels in the control of respiratory neuron discharge. Six protocols were used in this in vivo study of cVRG inspiratory (I) and expiratory (E) neurons in decerebrate, paralyzed, ventilated dogs. The protocols included characterizations of the neuronal responses to 1) BICm and apamin on the same neuron, 2) BICm during maximum apamin-induced block of AHPs, 3) apamin during maximum BICm-induced gain modulatory responses, 4) the specific GABA(A) receptor antagonist, (+)beta-hydrastine, 5) the specific GABA(A) receptor agonist, muscimol, and 6) the GABA uptake inhibitor, nipecotic acid. For protocols 3, 5, and 6, only E neurons were studied. Four-barrel micropipettes were used for extracellular single neuron recording and pressure ejection of drugs. Cycle-triggered histograms were used to quantify the F(n) patterns and to determine the drug-induced changes in the gain (slope) and offset of the F(n) patterns. Compared to apamin at maximum effective dose rates, BICm produced a 2.1-fold greater increase in peak F(n) and a 3.1-fold greater increase in average F(n). BICm and apamin produced similar increases in gain, but the offsets due to apamin were more negative. The responses to hydrastine were similar to BICm. During maximum apamin block, BICm produced an additional 112 +/- 22% increase in peak F(n). Conversely, apamin produced an additional 176 +/- 74% increase in peak F(n) during the maximum BICm-induced response. Muscimol and nipecotic acid both decreased the gain and offset of the discharge patterns. Taken together, these results suggest that the gain modulatory effect of BICm is due to a reduction of GABA(A)-ergic shunting inhibition rather than a reduction in AHPs by block of SK channels in canine cVRG neurons.

Mouse Mast Cell Secretory Granules Can Function as Intracellular Ionic Oscillators

Fluorescent Ca 2+ probes and digital photo-sectioning techniques were used to directly study the dynamics of Ca 2+ in isolated mast cell granules of normal (CB/J) and beige (Bg j/Bg j) mice. The resting intraluminal free Ca 2+ concentration ([Ca 2+] L) is 25 ± 4.2 μM (mean ± SD, n = 68). Exposure to 3 μM inositol 1,4,5-trisphosphate (InsP 3) induced periodic oscillations of luminal Ca 2+ ([Ca 2+] L) of ∼10 μM amplitude and a period around 8–10 s. The [Ca 2+] L oscillations were accompanied by a corresponding oscillatory release of [Ca 2+] L to the extraluminal space. Control experiments using ruthenium red (2 μM) and thapsigargin (100 nM) ruled out artifacts derived from the eventual presence of mitochondria or endoplasmic reticulum in the isolated granule preparation. Oscillations of [Ca 2+] L and Ca 2+ release result from a Ca 2+/K + exchange process whereby bound Ca is displaced from the heparin polyanionic matrix by inflow of K + into the granular lumen via an apamin-sensitive Ca 2+-sensitive K + channel (ASK Ca), whereas Ca 2+ release takes place via an InsP 3-receptor-Ca 2+ (InsP 3-R) channel. These results are consistent with previous observations of [Ca 2+] L oscillations and release in/from the endoplasmic reticulum and mucin granules, and suggest that a highly conserved common mechanism might be responsible for [Ca 2+] L oscillations and quantal periodic Ca 2+ release in/from intracellular Ca 2+ storage compartments.

Non-peptidic blockers of apamin-sensitive Ca 2+-activated K + channels

Non-peptidic blockers of apamin-sensitive Ca 2+-activated K + channels

Characterization of intestinal receptors for VIP and PACAP in rat and in PAC1 receptor knockout mouse.

The receptors for VIP and PACAP were characterized in vitro on rat ileal and colonic longitudinal smooth muscle with adherent myenteric ganglia. Colon strips from PAC1 receptor knockout and wildtype mice were also examined. VIP, PACAP-38 and PACAP-27 all caused concentration dependent relaxations. In rat ileum three different types of smooth muscle VIP/PACAP receptors were defined: (1) a PACAP-27 preferring receptor coupled to apamin sensitive Ca(2+)-dependent K+ channels, (2) a PAC1 receptor activated by both PACAP-27 and PACAP-38, and (3) a VIP specific receptor regulated by NPY. The receptors identified in rat colon were: (1) a PAC1 receptor localized on NO synthesizing neurones. Activation leads to increased NO production. (2) A smooth muscle PAC1 receptor. The responses elicited by both receptors were abolished by apamin. (3) A smooth muscle VIP specific receptor. PAC1 receptor knockout mice did not respond to PACAP-27 or PACAP-38, whereas VIP induced a relaxatory response indicating the presence of a VIP specific receptor. In wildtype mice all three peptides elicited relaxatory responses. Pharmacological characterization of intestinal VIP/PACAP receptors indicates the existence of receptors, such as a PACAP-27 preferring receptor and a VIP specific receptor, distinct from those that have been cloned (VPAC1, VPAC2, and PAC1).

Functional triads consisting of ryanodine receptors, Ca(2+) channels, and Ca(2+)-activated K(+) channels in bullfrog sympathetic neurons. Plastic modulation of action potential.

Fluorescent ryanodine revealed the distribution of ryanodine receptors in the submembrane cytoplasm (less than a few micrometers) of cultured bullfrog sympathetic ganglion cells. Rises in cytosolic Ca(2+) ([Ca(2+)](i)) elicited by single or repetitive action potentials (APs) propagated at a high speed (150 microm/s) in constant amplitude and rate of rise in the cytoplasm bearing ryanodine receptors, and then in the slower, waning manner in the deeper region. Ryanodine (10 microM), a ryanodine receptor blocker (and/or a half opener), or thapsigargin (1-2 microM), a Ca(2+)-pump blocker, or omega-conotoxin GVIA (omega-CgTx, 1 microM), a N-type Ca(2+) channel blocker, blocked the fast propagation, but did not affect the slower spread. Ca(2+) entry thus triggered the regenerative activation of Ca(2+)-induced Ca(2+) release (CICR) in the submembrane region, followed by buffered Ca(2+) diffusion in the deeper cytoplasm. Computer simulation assuming Ca(2+) release in the submembrane region reproduced the Ca(2+) dynamics. Ryanodine or thapsigargin decreased the rate of spike repolarization of an AP to 80%, but not in the presence of iberiotoxin (IbTx, 100 nM), a BK-type Ca(2+)-activated K(+) channel blocker, or omega-CgTx, both of which decreased the rate to 50%. The spike repolarization rate and the amplitude of a single AP-induced rise in [Ca(2+)](i) gradually decreased to a plateau during repetition of APs at 50 Hz, but reduced less in the presence of ryanodine or thapsigargin. The amplitude of each of the [Ca(2+)](i) rise correlated well with the reduction in the IbTx-sensitive component of spike repolarization. The apamin-sensitive SK-type Ca(2+)-activated K(+) current, underlying the afterhyperpolarization of APs, increased during repetitive APs, decayed faster than the accompanying rise in [Ca(2+)](i), and was suppressed by CICR blockers. Thus, ryanodine receptors form a functional triad with N-type Ca(2+) channels and BK channels, and a loose coupling with SK channels in bullfrog sympathetic neurons, plastically modulating AP.

Distinct mechanisms for activation of Cl- and K+ currents by Ca2+ from different sources in mouse sympathetic neurones.

We have investigated the roles of different voltage-dependent Ca2+ channels in the activation of the Cl- and K+ channels responsible for the afterdepolarization (ADP) and slow afterhyperpolarization (AHP) in sympathetic neurones of the isolated mouse superior cervical ganglion in vitro. The ADP and its associated Ca2+-activated Cl- current were markedly decreased by omega-agatoxin IVA (40-200 nM) and nifedipine (1-10 microM), but not by omega-conotoxin GVIA (300 nM). In contrast, the AHP and the apamin-sensitive Ca2+-activated K+ current that underlies this potential were blocked by omega-conotoxin GVIA, but were not affected by omega-agatoxin IVA and were only slightly reduced by nifedipine. Ryanodine (20 microM) reduced the Ca2+-activated Cl- current following an action potential by 75% but on average did not affect the Ca2+-activated K+ current. Evidence that R-type channels provide a proportion of the Ca2+ activating both types of Ca2+-dependent channel was obtained. We conclude that Ca2+ entering through L- and P-type Ca2+ channels preferentially activates the Cl- current responsible for the ADP in mouse sympathetic neurones, predominantly via Ca2+-induced Ca2+ release, whereas the Ca2+ that activates the K+ channels responsible for the AHP enters predominantly through N-type channels. The data can be explained by the selective association of each type of Ca2+ channel with particular intracellular mechanisms for activating other membrane channels, one indirect and the other direct, probably located at discrete sites on the soma and dendrites.

Bis-Quinolinium cyclophanes: 8,14-diaza-1,7(1, 4)-diquinolinacyclotetradecaphane (UCL 1848), a highly potent and selective, nonpeptidic blocker of the apamin-sensitive Ca(2+)-activated K(+) channel.

ADVERTISEMENT RETURN TO ISSUEPREVLetterNEXTBis-Quinolinium Cyclophanes: 8,14-Diaza-1,7(1,4)-diquinolinacyclotetradecaphane (UCL 1848), a Highly Potent and Selective, Nonpeptidic Blocker of the Apamin-Sensitive Ca2+-Activated K+ ChannelJian-Qing Chen, Dimitrios Galanakis, C. Robin Ganellin, Philip M. Dunn, and Donald H. JenkinsonView Author Information Departments of Chemistry and Pharmacology, University College London, Gower Street, London WC1E 6BT, U.K., and Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki, Thessaloniki 540 06, Greece Cite this: J. Med. Chem. 2000, 43, 19, 3478–3481Publication Date (Web):August 31, 2000Publication History Received10 January 2000Published online31 August 2000Published inissue 1 September 2000https://doi.org/10.1021/jm000904vCopyright © 2000 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views245Altmetric-Citations38LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (101 KB) Get e-AlertsSUBJECTS:Aromatic compounds,Hydrocarbons,Molecular structure,Particulate matter,Rodent models Get e-Alerts

The blocking effect of BmP02, one novel short-chain scorpion peptide on transient outward K + channel of adult rat ventricular myocyte

Two components F-2-7-4 and F-2-7-5, each composed of 28 amino acid residues, were purified from the venom of Buthus martensi Karsch by an opportune procedure with cation-exchange column chromatography and repeated HPLC. Both components were totally accounted to about 0.88% dry weight of the crude venom.The molecular weights of both components were determined to be 2950 and 2935 by mass spectrometry, which were fully coincidence with that of the known novel short-chain peptides BmP02 and BmP03, respectively [Romi-Lebrun R, Martin-Eauclaire M-F, Escoubas P, Wu FQ, Lebrun B, Hisada M, Nakajima T. Characterization of four toxins from Buthus martensi scorpion venom, which act on apamin-sensitive Ca 2+-activated K + channels. Eur J Biochem 1997;145:457–464]. In addition, the sequence of component F-2-7-4 was analyzed to be the same as that of BmP02. The components F-2-7-4 and F-2-7-5 purified in this study were, thus, finally distinguished to be BmP02 and BmP03 from the same venom. Using whole cell patch-clamp recording, it was found that BmP02 diminished the current of transient outward K + channel in adult rat ventricular myocyte in a concentration-dependent manner. The inhibitory effect was reversible. Dynamic studies showed that the activation, inactivation and recovery processes of the transient outward K + channel were not changed significantly after applying of BmP02. In addition, when BmP02 was applied to guinea pig ventricular myocyte, both delayed and inward rectified K + currents showed no change compared with the control. The results suggest strongly that BmP02 or -like peptides from scorpion venom may provide a useful probe for the studying of transient outward K + channel in rat ventricular myocyte.