Decrease in acetylcholine-induced current by neomycin in PC12 cells

The effects of neomycin, one of the aminoglycoside antibiotics, on the acetylcholine (ACh)-induced current (I(ACh)) were studied in pheochromocytoma cells by using the whole-cell clamp technique. The I(ACh) proved to be generated through neuronal nicotinic receptor. ACh (30 microM) induced an inward current at a holding potential of -80 mV. When cells were treated with neomycin (0.01-1 mM) and ACh (30 microM) simultaneously, an inhibitory effect of neomycin on the peak of I(ACh) was found. This effect was fast, reversible, and concentration dependent. Pretreatment with neomycin for 3-8 min had no effect on the inhibition of I(ACh) induced by neomycin. External application of 0.1 mM neomycin neither shifted the dose-response curve of the peak I(ACh) to the right (dissociation constant (K(d)) = 16.5 microM) nor affected its coefficient (1.8) but inhibited the curve amplitudes by approximately 33%. Stimulated protein kinase C activation by using an exogenous activator produced inhibition of I(ACh), while using protein kinase C inhibitor (PKCI 19-31) had no effect on the inhibition of I(ACh) induced by neomycin. These results suggest that neomycin has an inhibitory effect on I(ACh) without the involvement of phospholipase C. It indicates that neomycin binds to a specific site on the cell membrane, probably on the neuronal nicotinic receptor-coupled channel, and inhibits the I(ACh) in a noncompetitive manner, thus controlling the immediate catecholamine release from the sympathetic cells.     

Inhibitory effect of the kinase inhibitor chelerythrine on acetylcholine-induced current in PC12 cells.

In order to analyze the effect of protein kinase C(PKC) on nicotinic acetylcholine receptor in pheochromocytoma (PC12) cells by the whole-cell clamp technique, chelerythrine, a well-known inhibitor of PKC, was used to investigate the influence of PKC on acetylcholine (ACh)-induced current. When cells were preincubated with chelerythrine (0.1-10 microM) for 5 min, an inhibitory effect of chelerythrine on the peak of ACh-induced current was found. This effect was concentration-dependent, voltage-independent, and time-dependent within 1-6 min and reversible. However, intracellular dialysis with 0.1-5 microM PKCI 19-31, a specific pseudosubstrate PKC inhibitor, did not affect the inhibitory effect of chelerythrine. These results suggest that chelerythrine has an inhibitory effect on ACh-induced current in PC12 cells and that this effect is independent of its inhibition on PKC, may represent a new pharmacological effect of chelerythrine, and is mediated by an alternative mechanism.     

新霉素抑制PC12细胞烟碱受体介导的反应

新霉素是一种氨基甙类抗生素,在细胞水平可以抑制磷脂酶C介质的信号转导系统,本研究采用全细胞膜片钳技术,以大鼠肾上腺嗜铬细胞瘤细胞（PC12)为标本,观察了新霉素参考书国酰胆碱诱发电流（IACh)的影响,药理学鉴定表明,PC12细胞上的IACh是通过ACh激动烟碱受体引起的,钳制电压为－80mV时,ACh(30umol/L)诱发一内向电流;细胞外同时给予新霉素（0．01－1mmol/L)和ACh(30μmol/L)可显著抑制IACh峰值,此抑制作用迅速,可逆,呈浓度依赖性,用新霉素预处理细胞3－8min不影响其对IACh的抑制作用,用外源性蛋白激酶C（PKC）激剂激活PKC,同样可抑制IACh,而细胞内透析PKC抑制剂（PKCI19-31,0.1-5μmol/L）不影响新霉素对IACh的抑制作用,以上结果提示,新霉对PC12细胞的IACh的有抑制作用,这是一种与磷脂酶C阻断无关的药理学效应。     

糖皮质激素对大鼠肾上腺髓质嗜铬细胞瘤细胞ACh诱发电流的快速抑制作用

本研究采用全细胞膜片箝 技术,以大鼠肾上腺髓质嗜铬细胞瘤细胞为标本,观察了糖皮质激素对乙胆碱诱发电流的快速作用,并初步探讨了其可能机制。     

The inhibitory and facilitatory actions of amyloid-beta peptides on nicotinic ACh receptors and AMPA receptors

The present study investigated the effects of amyloid-beta peptides on nicotinic ACh receptors (Torpedo, alpha 4 beta 2, and alpha 7 receptors) and AMPA receptors expressed in Xenopus oocytes by monitoring whole-cell membrane currents. Ten-minutes treatment with amyloid-beta(1-42) (1 microM) inhibited Torpedo ACh receptor currents, reaching 53% of original levels 30 min after treatment. Amyloid-beta(1-40) inhibited the currents in a dose-dependent manner (0.1-10 microM) during treatment, gradually reversing after treatment. Amyloid-beta(1-40) and amyloid-beta(1-42) (0.1 microM) depressed alpha 4 beta 2 receptor currents to each 69% and 62% of original levels at 10-min treatment and lesser depression was obtained with alpha 7 receptors. Amyloid-beta(1-42) (0.1 microM) did not significantly inhibit AMPA receptor currents, but amyloid-beta(1-40) (0.1 microM) potentiated the currents to 145-191% of original levels. Amyloid-beta peptides, thus, exert their diverse actions on nicotinic ACh receptors and AMPA receptors, and the inhibitory actions on nicotinic ACh receptors may account for the deterioration of learning and memory in Alzheimer's disease.     

Suppression of the Nicotinic Acetylcholine Response in Rat Superior Cervical Ganglionic Neurons by Steroids

Abstract : The effects of various types of steroids on the nicotinic acetylcholine (ACh) receptor (nAChR)-mediated responses were investigated in superior cervical ganglionic neurons acutely dissociated from rats using nystatin perforated patch recording. ACh induced a peak followed by a gradual decrease in the inward current at a holding potential of -40 mV. Nicotine, but not muscarine, mimicked ACh. Hydrocortisone at a concentration of > 10^-6 M reversibly suppressed both the peak and steady-state nicotine-induced currents ( I _nic) in a noncompetitive manner. The inhibition of I _nic by hydrocortisone did not show any voltage dependency and persisted in the presence of either cyclic AMP modulators, forskolin and 3-isobutyl-1-methylxanthine, or a protein kinase A inhibitor, N -[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89). β-Estradiol, androsterone, aldosterone, and 17α-estradiol mimicked hydrocortisone in its inhibitory action on ACh-induced currents ( I _ACh). The potency for the inhibitory actions on I _Ach was as follows : androsterne > β-estradiol > hydrocortisone ≥ aldosterone =17α-estradiol. Cholesterol had no effect on the I _ACh. In conclusion, the structural characteristics of steroid are thus considered to be necessary to block nicotinic I _ACh in rat superior cervical ganglionic cells, whereas the cholesterol side chain might disturb the inhibitory action of the steroid skeleton on nAChRs.     

Nicotinic autoreceptor function in rat brain during maturation and aging: possible differential sensitivity to organophosphorus anticholinesterases

Acetylcholine (ACh) release is modulated pre-synaptically by both muscarinic and nicotinic receptor-mediated processes. While muscarinic autoreceptors inhibit ACh release, nicotinic autoreceptors enhance ACh release and thus disruption of these processes could potentially affect cholinergic toxicity following exposure to anticholinesterases. Marked age-related differences in sensitivity to some organophosphorus (OP) anticholinesterases have been reported. We compared nicotinic autoreceptor function (NAF) during maturation and aging and evaluated its potential modulation by the common OP insecticide, chlorpyrifos (CPF). Cortical synaptosomes were pre-loaded with [3H]choline, superfused (0.6 ml/min) with physiological buffer and [3H]ACh release was evoked with potassium (KCl, 9 mM), with or without co-addition of exogenous ACh to stimulate nicotinic autoreceptors. Fractions of perfusate were subsequently collected and area under the curve (AUC) for [3H] was analyzed by scintillation counting. The difference in evoked release due to co-addition of exogenous ACh was defined as NAF. Under these conditions, atropine (ATR, 0.1 microM) appeared requisite for NAF; thus this muscarinic antagonist was subsequently added to all perfusion buffers. In synaptosomes from adult tissues, exogenous ACh (3-100 microM) significantly increased release in a concentration-dependent manner. The nicotinic antagonist mecamylamine (MEC, 100 microM) substantially reduced the potassium-evoked release elicited by co-addition of ACh (10 microM). Interestingly, the nicotinic agonists nicotine (NIC) and dimethylphenylpiperazinium (DMPP; 0.1-10 microM) had no effect on release. The active metabolite of CPF (i.e. chlorpyrifos oxon (CPO), 1-10 microM) inhibited NAF in vitro. Maturation-related expression of NAF was noted (AUC with co-addition of 10 microM ACh: 7-day rats, 7+/-6; 21-day rats, 44+/-6; 90-day rats, 196+/-37; 24-month rats, 173+/-52). NAF was substantially reduced (67-91%) 96 h after maximum tolerated dosages of CPF in adult and aged rats (279 mg/kg, sc) but not in juveniles (127 mg/kg, sc), even though AChE inhibition was similar among the age groups (>80%). Together these data suggest that NAF is differentially expressed during maturation and that this neuromodulatory process may be selectively altered by some OP insecticides, potentially contributing to age-related differences in response to AChE inhibitors. As NAF has been postulated to be activated under conditions of 'impaired' cholinergic function, selective alteration of this pre-synaptic process by OP anticholinesterases may be also important in age-related conditions associated with cholinergic hypofunction.     

ACh对大鼠皮层体感区神经元延迟整流钾电流的抑制作用

利用全细胞膜片钳技术研究乙酰胆碱(acetylcholine,ACh)对大鼠皮层体感区神经元延迟整流钾电流(IK)的调制作用。结果表明:(1)ACh(0．1、1、10、100 μmol/L)对大鼠皮层体感区神经元IK有抑制作用,并具有剂量依赖性关系(P<0．01)。 (2)ACh可使IK激活曲线的斜率变大,并使激活曲线向超极化方向移动。IK激活曲线的半数激活电压(V1/12)和斜率因子(k)分别由给药前的(-41．8±9．7)mV和(30．7±7．2)mV变为给药后的(-122．4±38．6)mV和(42．4±7．0)mV。(3)100 μmol/L的N受体拮抗剂筒箭毒碱(tubocurarine)可减弱ACh对IK的抑制作用,在指令电压+60 mV时tubocurarine+ACh组的IK幅度下降了(16．9± 13．8)％(n=8),与10 μmol/L ACh组引起的(36．5±7．8)％的IK下降幅度相比,有极显著差异(P<0．01)。10 μmol/L的M1受体拮抗剂哌仑西平(pirenzepin)拮抗ACh对IK的抑制作用不明显(n=7,P>0．05);而10 μmol/L的M3受体拮抗剂4-DAMP可部分拮抗ACh对IK的抑制作用,并且4-DAMP+ACh组使IK的电流值下降了(26．8±4．7)％(n=6),与ACh组引起的IK电流下降相比,有显著差异(P<0．05)。(4)蛋白激酶C(protein kinase C,PKC)阻断剂chelerythrine拮抗ACh对IK的抑制作用,PKC激动剂PDBu可增强ACh对IK的抑制作用(P<0．05)。综上所述,ACh对人鼠皮层体感区神经元IK的抑制作用主要是通过烟碱受体(nAChRs)和M3受体介导,并经过PKC信号途径。     

The effect of C-type natriuretic peptide on delayed rectifier potassium currents in gastric antral circular myocytes of the guinea-pig

C-type natriuretic peptides (CNP) play an inhibitory role in smooth muscle motility of the gastrointestinal tract, but the effect of CNP on delayed rectifier potassium currents is still unclear. This study was designed to investigate the effect of CNP on delayed rectifier potassium currents and its mechanism by using conventional whole-cell patch-clamp technique in guinea-pig gastric myocytes isolated by collagenase. CNP significantly inhibited delayed rectifier potassium currents [I(K (V))] in dose-dependent manner, and CNP inhibited the peak current elicited by depolarized step pulse to 86.1+/-1.6 % (n=7, P<0.05), 78.4+/-2.6 % (n=10, P<0.01) and 67.7+/-2.3 % (n=14, P<0.01), at concentrations of 0.01 micromol/l, 0.1 micromol/l and 1 micromol/l, respectively, at +60 mV. When the cells were preincubated with 0.1 micromol/l LY83583, a guanylate cyclase inhibitor, the 1 ?micromol/l CNP-induced inhibition of I(K (V)) was significantly impaired but when the cells were preincubated with 0.1 micromol/l zaprinast, a cGMP-sensitive phosphodiesterase inhibitor, the 0.01 micromol/l CNP-induced inhibition of I(K (V)) was significantly potentiated. 8-Br-cGMP, a membrane permeable cGMP analogue mimicked inhibitory effect of CNP on I(K (V)). CNP-induced inhibition of I(K (V)) was completely blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). The results suggest that CNP inhibits the delayed rectifier potassium currents via cGMP-PKG signal pathway in the gastric antral circular myocytes of the guinea-pig.     

The protein kinase inhibitor, staurosporine, inhibits L-type Ca2+ current in rabbit atrial myocytes

A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca(2+) channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I-V relationship of LTCC (from -16.8+/-2.55 to -3.74+/-1.22pApF(-1) at 0 mV). The level of L-type Ca(2+) current (I(Ca,L)) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I(Ca,L) in a dose-dependent manner with a K(d) value of 61.98+/-6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 microM) and chelerythrine (3 microM), and PKA inhibitor, PKA-IP (5 microM), did not show any inhibitory effect on I(Ca,L). Additional application of ST in the presence of isoproterenol (1 microM), a selective beta-adrenoreceptor agonist, reduced peak I(Ca,L) (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.     

Selected contribution: effect of the aldehyde acrolein on acetylcholine-induced membrane current in airway smooth muscle cells.

Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca(2+) signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at -60 mV, ACh (0.1-10 microM) induced a concentration-dependent inward current, which, in approximately 50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 microM). Exposure to acrolein (0.2 microM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 microM) concentration-induced initial peak of current (318.8 +/- 28.3 vs. 251.2 +/- 40.3 pA; n = 25, P < 0.05). At a high-ACh concentration (10 microM), the frequency at which subsequent peaks occurred was significantly increased (13.2 +/- 1.1 vs. 8.7 +/- 2 min(-1); n = 20, P < 0.05). ACh-induced current was identified as a Ca(2+)-activated Cl(-) current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca(2+) release, did not alter caffeine-induced transient membrane currents (595 +/- 45 and 640 +/- 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca(2+) concentration response and that the protective role of inhibitors of Cl(-) channels in air pollutant-induced airway hyperresponsiveness should be examined.     

The Inhibitory Effect of Trifluoromethylphenylpiperazine on [3H]Acetylcholine Release in Guinea Pig Hippocampal Synaptosomes Is Mediated by a 5-Hydroxytryptamine1 Receptor Distinct from 1A, 1B, and 1C Subtypes

The effect of the serotonergic receptor agonist 1-(m-trifluoromethylphenyl)piperazine (TFMPP) was studied on the K(+)-evoked [3H]acetylcholine [( 3H]ACh) release from guinea pig hippocampal synaptosomes loaded with [3H]choline. TFMPP (5-1,000 microM) inhibited the evoked ACh release in a dose-dependent manner (IC50 = 81.8 microM). The inhibitory effect of TFMPP was mimicked by CGS-12066B (10, 30, and 100 microM), a 5-hydroxytryptamine1B (5-HT1B)/5-HT1D receptor agonist; 1-(m-chlorophenyl)piperazine (100 microM), a 5-HT1C/5-HT1B receptor agonist; and 5-carboxamidotryptamine (10 microM), a nonselective 5-HT1 receptor agonist. 8-Hydroxy-2-(di-n-propylamino)tetralin (10 and 100 microM), a 5-HT1A receptor agonist, and quipazine (10 and 100 microM), a 5-HT2 receptor agonist, did not have any significant effect. Serotonergic antagonists, such as dihydroergotamine (0.1 and 1 microM), metergoline (0.1 microM), methysergide (0.5 and 1 microM), or yohimbine (1 and 10 microM), blocked the TFMPP effect dose-dependently. In contrast, methiotepine (0.3 and 1 microM), propranolol (1 microM), ketanserin (0.1 microM), mesulergine (0.1 microM), ICS 205930 (0.1 and 1 microM), and spiroperidol (1 and 7 microM) did not affect the TFMPP-induced inhibition of the evoked ACh release. These data suggest that, in guinea pig hippocampus, the K(+)-evoked ACh release is modulated by a 5-HT1 receptor distinct from the 5-HT1A, 5-HT1B, and 5-HT1C subtypes.     

The role of ether-a-go-go-related gene K(+) channels in glucocorticoid inhibition of adrenocorticotropin release by rat pituitary cells

The present study investigated the role of K(+) channels in the inhibitory effect of glucocorticoid on adrenocorticotropin (ACTH) release induced by corticotropin-releasing hormone (CRH) using cultured rat anterior pituitary cells. Apamin and charybdotoxin (CTX) did not have a significant effect on ACTH release induced by CRH (1 nM). Tetraethylammonium (TEA), a broad spectrum K(+) channel blocker, increased the ACTH response to CRH only at the highest concentration (10 mM). The exposure to 100 nM corticosterone for 60 min inhibited the CRH-induced ACTH release. Neither TEA, apamin, nor CTX affected the inhibitory effect of corticosterone. In contrast, astemizole (Ast) and E-4031, ether-a-go-go-related gene (erg) K(+) channel blockers, abolished the inhibitory effect of corticosterone on CRH-induced ACTH release (1.25+/-0.10 vs. 1.45+/-0.11 ng/well at 10 microM Ast, p>0.05, 1.71+/-0.16 vs. 1.91+/-0.32 ng/well at 10 microM E-4031, p>0.05, vehicle vs. corticosterone). RT-PCR demonstrated all three subtypes of rat-erg mRNAs in the pituitary and corticosterone increased only erg1 mRNA up to 2.47+/-0.54 fold. In conclusion, erg K(+) channels were up-regulated by glucocorticoid, and have indispensable roles in delayed glucocorticoid inhibition of CRH-induced ACTH release by rat pituitary cells.     

Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl(-) currents (I(Cl.vol)) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.1 microM) or by hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 microM) or Ro-31-8425 (0.1 microM), inhibitors of both conventional and novel PKC isozymes, significantly activated I(Cl.vol) and prevented further modulation by subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 microM), a selective conventional PKC inhibitor, was without effect. Dialyzing acutely dispersed and cultured PASMC with epsilon V1-2 (10 microM), a translocation inhibitory peptide derived from the V1 region of epsilon PKC, activated I(Cl.vol) under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with beta C2-2 (10 microM), a translocation inhibitory peptide derived from the C2 region of beta PKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of epsilon PKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound epsilon PKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.     

Effect of NYD-SP27 down-regulation on ATP-induced Ca2+-dependent pancreatic duct anion secretion in cystic fibrosis cells

Our previous study demonstrated that NYD-SP27 is a novel inhibitory PLC isoform expressed endogenously in human pancreas and upregulated in CFPAC-1 cells. The present study investigated the effect of NYD-SP27 down-regulation on the ATP-stimulated and Ca(2+)-dependent pancreatic anion secretion by CFPAC-1 cell line using short-circuit current (I(SC)) recording. NYD-SP27 antisense-transfected CFPAC-1 (AT-CF) cells exhibited a significantly higher basal transmembrane potential difference and current than those of empty vector-transfected CFPAC-1 (VT-CF) cells. Cl(-) channel blocker, DPC or Glibenclamide (1mM), and inhibitor of Na(+)-K(+)-Cl(-) cotransporter, bumetanide (100 microM) significantly inhibited the basal current in AT-CF cells. The inhibitor of adenylate cyclase, MDL12330A (20 microM), and Ca(2+)-dependent Cl(-) channel (CaCC) blocker, DIDS (100 microM) also significantly reduced the basal current in AT-CF. Apical application of ATP (10 microM) stimulated a fast transient I(SC) increase in VT-CF cells, but a more sustained rise with slower decline in AT-CF cells. Pretreatment with BAPTA-AM (50 microM) reduced the ATP-induced I(SC) response in AT-CF cells by 77.9%. PMA (1 microM), a PKC activator, inhibited the ATP-stimulated current increase (the transient peak) in VT-CF cells, but had no effect on the AT-CF cells. However, PKC inhibitor, staurosporine (40 microM) could inhibit the ATP-induced I(SC) response in AT-CF cells. The present results confirm the previously proposed inhibitory role of NYD-SP27 in the PLC pathway and demonstrate that the suppression of its expression could result in an enhancement of ATP-stimulated Ca(2+) dependent pancreatic anion secretion.     

A hydrophobic photolabel inhibits nicotinic acetylcholine receptors via open-channel block following a slow step.

3-(Trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) is a hydrophobic inhibitor of nicotinic acetylcholine receptors (nAChRs) and a photolabel that incorporates both at the lipid-protein interface and within the gated pore. On the basis of Torpedo vesicle studies, TID is thought to selectively inhibit the closed nAChR state. The nAChR site(s) mediating TID inhibition is unknown. We investigated the state dependence and kinetics of TID inhibition electrophysiologically using rapidly superfused membrane patches expressing mouse muscle nAChRs. Currents from patches simultaneously exposed to ACh and TID show no inhibition of peak currents relative to acetylcholine (ACh) alone but demonstrate slow (10 s(-1)) TID inhibition. Patch preexposure to TID before ACh results in a burst of current followed by rapid [TID]-dependent inhibition at a bimolecular rate of 1.8 x 10(8) M(-1) s(-1), indicating that TID selectively inhibits open channels. We also determined sensitivity to TID in two nAChRs containing mutations in their pore-forming M2 domains. The alphaL251T mutation eliminates sensitivity to TID inhibition, while the alphaS252I mutation enhances this sensitivity 4-fold compared to wild type. These results indicate that TID inhibition of nAChRs follows two distinct kinetic steps. The rate-limiting step, which shows features suggesting a diffusion barrier, precedes rapid open-state-dependent TID binding to an inhibition site near the putative nAChR gate.     

Arachidonic acid modulation of alpha1H, a cloned human T-type calcium channel

Arachidonic acid (AA) and the products of its metabolism are central mediators of changes in cellular excitability. We show that the recently cloned and expressed T-type or low-voltage-activated Ca channel, alpha1H, is modulated by external AA. AA (10 microM) causes a slow, time-dependent attenuation of alpha1H current. At a holding potential of -80 mV, 10 microM AA reduces peak inward alpha1H current by 15% in 15 min and 70% in 30 min and shifts the steady-state inactivation curve -25 mV. AA inhibition was not affected by applying the cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor nordihydroguaiaretic acid. The epoxygenase inhibitor octadecynoic acid partially antagonized AA attenuation of alpha1H. The epoxygenase metabolite epoxyeicosatrienoic acid (8,9-EET) mimicked the inhibitory effect of AA on alpha1H peak current. A protein kinase C (PKC)-specific inhibitor (peptide fragment 19-36) only partially antagonized the AA-induced reduction of peak alpha1H current and the shift of the steady-state inactivation curve but had no effect on 8,9-EET-induced attenuation of current. In contrast, PKA has no role in the modulation of alpha1H. These results suggest that AA attenuation and shift of alpha1H may be mediated directly by AA. The heterologous expression of T-type Ca channels allows us to study for the first time properties of this important class of ion channel in isolation. There is a significant overlap of the steady-state activation and inactivation curves, which implies a substantial window current. The selective shift of the steady-state inactivation curve by AA reduces peak Ca current and eliminates the window current. We conclude that AA may partly mediate physiological effects such as vasodilatation via the attenuation of T-type Ca channel current and the elimination of a T-type channel steady window current.     

The K(+)-evoked release of [3H]acetylcholine from slices of rat globus pallidus: modulation by delta-opioid receptors.

Previous investigations have shown that the activation of delta-opioid receptors depresses the release of acetylcholine (ACh) in the rat caudate putamen. This finding raised the possibility that the release of ACh is similarly modulated in the globus pallidus, a region containing a distinct population of cholinergic neurons and enriched in enkephalinergic nerve terminals. In the present study the pallidal release of ACh was characterized and the effects of delta-opioid receptor activation on this release were examined. The results show that this release is stimulated by high K+ in a concentration- and Ca(2+)-dependent manner. D-Pen2,L-Pen5-enkephalin (0.1-10 microM), a selective delta-opioid receptor agonist, produced a dose-related inhibition of the 25 mM K(+)-evoked tritium release. The maximal inhibitory effect, representing a 34% decrease in the K(+)-induced tritium release, was observed at a concentration of 1 microM. This opioid effect was attenuated by the selective delta-opioid receptor antagonist, ICI 174864 (1 microM). These findings support the role of a delta-opioid receptor in the modulation of ACh release in the rat globus pallidus.     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

Endothelium-dependent vasodilation in different rat hindlimb skeletal muscles.

Few studies have examined potential for endothelium-dependent vasodilation in skeletal muscles of different fiber-type composition. We hypothesized that muscles composed of slow oxidative (SO)- and/or fast oxidative glycolytic (FOG)-type fibers have greater potential for endothelium-dependent vasodilation than muscles composed of fast glycolytic (FG)-type fibers. To test this hypothesis, the isolated perfused rat hindlimb preparation was used with a constant-flow, variable-pressure approach. Perfusion pressure was monitored continuously, and muscle-specific flows were determined by using radiolabeled microspheres at four time points: control, at peak effect of acetylcholine (ACh I; 1-2 x 10(-4) M), at peak effect of ACh after infusion of an endothelial inhibitor (ACh II), and at peak effect of sodium nitroprusside (SNP; 4-5 x 10(-4) M). Conductance was calculated by using pressure and flow data. In the SO-type soleus muscle, conductance increased with ACh and SNP, but the increase in conductance with ACh was partially abolished by the endothelial inhibitor N(G)-nitro-l-arginine methyl ester (control, 0.87 +/- 0.19; ACh I, 2.07 +/- 0.29; ACh II, 1.32 +/- 0.15; SNP, 1.76 +/- 0.19 ml. min(-1). 100 g(-1). mmHg(-1); P < 0.05, ACh I and SNP vs. control). In the FOG-type red gastrocnemius muscle, similar findings were obtained (control, 0.64 +/- 0.11; ACh I, 1.36 +/- 0.21; ACh II, 0.73 +/- 0.16; SNP, 1.30 +/- 0.21 ml. min(-1). 100 g(-1). mmHg; P < 0.05, ACh I and SNP vs. control). In the FG-type white gastrocnemius muscle, neither ACh nor SNP increased conductance. Similar findings were obtained when muscles were combined into high- and low-oxidative muscle groups. Indomethacin had no effect on responses to ACh. These data indicate that endothelium-dependent vasodilation is exhibited by high-oxidative, but not low-oxidative, rat skeletal muscle. Furthermore, endothelium-dependent vasodilation in high-oxidative muscle appears to be primarily mediated by nitric oxide.