Differential age-dependent expression of α2 adrenoceptor- and P2 purinoceptor-functions in rat locus coeruleus neurons

Whole-cell patch clamp recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). In a first series of experiments, it was demonstrated that tyrosine hydroxylase-positive LC neurons of young (10–14 days of age) rats are multipolar with numerous dendrites. When pipettes filled with the marker molecule biocytin were used for recording, all cells exhibiting outward current responses to noradrenaline (100 μM) showed the morphology typical for LC neurons. At a holding potential of –80 mV, noradrenaline (100 μM) produced a comparably small outward current both in LC neurons of young (8–14 days) and older (18–23 days) rats. In contrast, 2-methylthio ATP (2-MeSATP; 100 μM) caused a relatively small inward current in the young animals, while inward current responses were much larger in most older animals (8 out of a total of 11). It is suggested that after birth there are probably no functional P2 purinoceptors present at LC neurons. Thereafter, P2 purinoceptor-function increases with age, reaching maturity only in animals older than 18 days. Received: 10 October 1997 / Accepted: 4 November 1997     

Comparison of the effects of nitric oxide synthase inhibition and guanylate cyclase inhibition on vascular contraction in vitro and in vivo in the rat

We have investigated the differences between the nitric oxide synthase inhibitor (NOSI), L-NMMA, and the guanylate cyclase inhibitors (GCI), methylene blue and LY 83583, in their abilities to increase vasoconstrictor responses in vitro and in vivo. In rat small mesenteric arterial rings, 1 h exposure to the NOSI, L-NMMA (100 μM), and the GCI, methylene blue (10 μM), alone or in combination with L-NMMA, caused a significant reduction in the maximum relaxation to ACh in mesenteric arteries pre-contracted with the thromboxane mimetic U46619 (10 μM). Hence, both NOSI and GCI inhibit endothelium-dependent relaxations to ACh in rat small mesenteric artery. However, 1 h exposure to L-NMMA and L-NNA (both 100 μM), but not methylene blue (10 μM), significantly increased the contractile response to U-46619 (10 μM) in rat small mesenteric artery. It was decided to investigate further this difference between NOSI and methylene blue. In rat small mesenteric arterial rings, L-NMMA (10 μM) and LY 83583 (1–10 μM) significantly increased the contractile response to KCl (40 mM) or to noradrenaline (10 μM), when administered during the contraction. However, methylene blue (1–10 μM) increased the contractile response to KCl but not noradrenaline. In rat aortic rings, L-NMMA (100 μM), methylene blue (1–10 μM) and LY 83583 (1–10 μM) significantly increased the contractile response to KCl (40 mM) or to noradrenaline (1 μM). In the pithed rat preparation, L-NMMA (40.3 μmol kg^–1, i.v.) significantly increased the pressor response both to bolus injection of noradrenaline (3.13 nmol kg^–1) and to spinal pressor nerve stimulation. However, methylene blue (3.13–15.6 μmol kg^–1) or LY 83583 (4.0–40.0 μmol kg^–1), failed to affect pressor responses to either NA or pressor nerve stimulation. Hence, there are differences between NOSI and GCI in their abilities to increase vasoconstrictor responses, especially when comparing responses in vitro and in vivo. This suggests that nitric oxide has actions in addition to activation of guanylate cyclase to modulate vasoconstrictor responses, presumably by membrane hyperpolarisation, and that this action may be more important in vivo. Received: 2 May 1997 / Accepted: 19 June 1997     

Evidence for the involvement of multiple mechanisms in the excitatory action of bradykinin in the circular muscle of guinea-pig colon

We have investigated, by using the sucrose gap technique, the mechanisms of the excitatory action of bradykinin in the circular muscle of the guinea-pig proximal colon. In the presence of atropine (1μM) and S-ketoprofen (3μM), the application of bradykinin (1μM for 20s) produced complex changes in membrane potential and muscle tension. The prevailing response was a small hyperpolarization followed by a slowly developing depolarization and a tonic contraction. The selective B₂ receptor antagonist, HOE 140 (0.3μM) blocked the responses to bradykinin (1μM) while tetrodotoxin (0.3μM) had no affect. The selective B₁ receptor agonist, [des-Arg⁹]bradykinin (1μM) did not affect the electrical or mechanical activities of the circular muscle. Apamin (0.1μM) blocked the transient hyperpolarization and potentiated the bradykinin-induced depolarization and contraction. In the presence of apamin, nifedipine (1μM) blocked spikes (when present) and the phasic contraction while leaving the tonic contraction unaffected. The excitatory action of bradykinin was further investigated in the presence of atropine (1μM), S-ketoprofen (3μM), apamin (0.1μM) and nifedipine (1μM). The depolarization but not the contraction induced by bradykinin was reduced by about 30% in low-Na⁺ (25mM) but not in low Cl^– (9.7mM) Krebs solution. The depolarization and contraction evoked by bradykinin were reduced (by about 30 and 75%, respectively) in Ca²⁺-free (2min) Krebs solution. The blocker of the sarcoplasmic reticulum Ca²⁺ pump, cyclopiazonic acid (CPA, 10μM) reduced the nifedipine-resistant depolarization and contraction induced by bradykinin by about 40 and 60%, respectively. The inhibitor of receptor-operated cation channels, SKF 96365 (50μM) reduced the nifedipine-resistant bradykinin-induced depolarization and contraction by about 40 and 30%, respectively, whereas the inhibitor of Ca²⁺-dependent chloride channels, niflumic acid (100μM) was without effect. The inhibitory effect of SKF 96365 (50μM) and CPA (10μM) was additive: in the presence of both drugs the bradykinin-induced depolarization and contraction were reduced by about 70–80%. The proteine kinase C inhibitor, GF 109203x (10μM) did not affect the nifedipine-resistant bradykinin-induced depolarization and contraction. At a concentration of 30μM, GF 109203x reduced the bradykinin-induced contraction by about 50% while leaving the bradykinin-induced depolarization unaffected. The KCl (40mM)-induced contraction was significantly reduced (by about 30%) by GF 109203x (30μM). The present findings indicate that, in the presence of apamin and nifedipine, the bradykinin-induced contraction of circular muscle of the guinea-pig colon is due to the influx of extracellular Ca²⁺ via non-selective cation channels and, in part, to the release of Ca²⁺ from a loosely bound internal store. Intracellular Ca²⁺ facilitates the bradykinin-induced depolarization, a response which does not involve a protein kinase C-dependent mechanism. Received: 4 August 1997 / Accepted: 26 November 1997     

Effects of duramycin on cardiac voltage-gated ion channels

The amphipathic peptide duramycin is in clinical development for the treatment of cystic fibrosis. It is deposited in cellular membranes where it binds to phosphatidylethanolamine. Duramycin may thereby change the biophysical membrane properties and perturb the function of ion channels. If so, in heart tissue, its application carries the risk to elicit cardiac arrhythmias. In fact, premature ventricular complexes were observed in the electrocardiogram during toxicological testing in dogs. To study the arrhythmogenic potential of duramycin, we investigated its effects on currents through voltage-gated hERG potassium, sodium, and calcium channels in native cells, and using a heterologous expression system, by means of the whole-cell patch clamp technique; duramycin bath concentrations between 1 nM and 0.1 μM did not generate any effects on these currents. Concentrations ≥0.3 μM, however, reduced the amplitudes of all investigated currents. Moreover, sodium current fast inactivation kinetics was slowed in the presence of duramycin. A further rise in duramycin bath concentration (≥3.3 μM) induced a leak current consistent with pore formation. The reported effects of duramycin on ion channel function are likely to arise from a change in the biophysical properties of the membrane rather than from a specific interaction of the peptide with ion channel proteins. Under therapeutic conditions (i.e., administration via inhalation), duramycin plasma concentrations are below 0.5 nM. Thus, upon inhalation, duramycin has a large safety margin and is highly unlikely to elicit arrhythmias. Eva Zebedin, and Xaver Koenig contributed equally.     

Modulation of serotonin-induced currents by metals in mouse neuroblastoma cells

 The effects of several metals on the serotonin receptor-channel complex were studied using mouse neuroblastoma N1E-115 cells which are known to be endowed with the 5-HT₃ subclass of the receptor. The whole-cell patch clamp technique was used to record currents induced by serotonin at a concentration of 3 μM which was equivalent to the apparent dissociation constant. Methylmercury and mercuric chloride suppressed serotonin-induced currents irreversibly, with a 50% suppression being observed at concentrations of 3 μM and 2 μM, respectively. Lead and zinc suppressed the current with IC₅₀s of 80 μM and 50 μM, respectively, and the effects of both metals were reversible after washing with metal-free solution. Lanthanum also suppressed the current with an IC₅₀ of 10 μM, and the effect was partially reversible. Cadmium and cobalt augmented serotonin-induced currents slightly but consistently at a concentration of 100 μM, and the effect was reversible. Aluminum at 100 μM had no effect on serotonin-induced currents. It was concluded that the 5-HT₃ receptor is endowed with a unique property with respect to the actions of metals which is not shared by some other ligand-gated and voltage-gated ion channels. Received: 15 November 1995/Accepted: 18 January 1996     

Staurosporine directly blocks Kv1.3 channels expressed in Chinese hamster ovary cells

The effects of staurosporine (ST), a widely used protein kinase C (PKC) inhibitor, were examined on Kv1.3 channels stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell and excised inside-out configurations of the patch clamp technique. In whole-cell recordings, ST, at external concentrations from 300 nM to 10 μM, accelerated the rate of inactivation of Kv1.3 currents and thereby reduced the current at the end of the depolarizing pulse in a concentration-dependent manner with an IC₅₀ of 1.2 μM. The actions of ST were unaffected by pretreatment with another selective PKC inhibitor, chelerythrine, or by including the PKC pseudosubstrate peptide inhibitor, PKC 19-36, in the intracellular solution. Rp-cAMPS, a specific protein kinase A inhibitor, included in intracellular solution did not affect the effects of ST. Furthermore, the same effects of ST on Kv1.3 were also observed in excised inside-out patches when applied to the internal face of the membrane. These effects were completely reversible upon washing. Current-voltage relations for Kv1.3 currents at the end of voltage steps indicated that ST reduced Kv1.3 currents over a wide voltage range. The blockade exhibited a shallow voltage dependence between –10 mV and +40 mV, increasing at more positive potentials. ST had no effect on the voltage dependence of steady-state inactivation. It reduced the tail current amplitude and slowed the deactivation time course, resulting in a crossover phenomenon. These results suggest that the action of ST on Kv1.3 is independent of PKC and PKA inhibition. ST blocks the open state of Kv1.3 channels to produce an apparent acceleration of the inactivation rate. Received: 19 October 1998 / Accepted: 12 January 1999     

The anti-nociceptive agent ralfinamide inhibits tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ currents in dorsal root ganglion neurons

Tetrodotoxin-resistant and tetrodotoxin-sensitive Na⁺ channels contribute to the abnormal spontaneous firing in dorsal root ganglion neurons associated with neuropathic pain. Effects of the anti-nociceptive agent ralfinamide on tetrodotoxin-resistant and tetrodotoxin-sensitive currents in rat dorsal root ganglion neurons were therefore investigated by patch clamp experiments. Ralfinamide inhibition was voltage-dependent showing highest potency towards inactivated channels. IC₅₀ values for tonic block of half-maximal inactivated tetrodotoxin-resistant and tetrodotoxin-sensitive currents were 10 μM and 22 μM. Carbamazepine, an anticonvulsant used in the treatment of pain, showed significantly lower potency. Ralfinamide produced a hyperpolarising shift in the steady-state inactivation curves of both currents confirming the preferential interaction with inactivated channels. Additionally, ralfinamide use and frequency dependently inhibited both currents and significantly delayed repriming from inactivation. All effects were more pronounced for tetrodotoxin-resistant than tetrodotoxin-sensitive currents. The potency and mechanisms of actions of ralfinamide provide a hypothesis for the anti-nociceptive properties found in animal models.     

Inhibitory effect of 1,1,1-trichloroethane on calcium channels of neurons

Peripheral neuropathy may remain for some time after 1,1,1-trichloroethane exposure. A variety of Ca2+ channels gives sensory neurons many kinds of transmitting sensory information. We measured calcium currents in sensory neurons from neonatal rat dorsal root ganglion using whole-cell patch-clamp recordings. Trichloroethane reversibly reduced the low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium. The half-inhibitory concentration (IC50) of the HVA and LVA currents was 5.76 x 10(-3) M and 3.99 x 10(-3) M, respectively. The Hill coefficient of the HVA and LVA currents was 0.61 and 1.04, respectively. In assessing voltage dependence for activation and inactivation of calcium currents, only the HVA calcium current was inactivated at greater negative potentials. This may be one of the mechanisms to reduce HVA current. However, activation and inactivation of the LVA currents were not affected by trichlorothane, so inhibition of the LVA currents may have other mechanisms. Calcium currents are thought to be involved in the control of neuronal excitability and neurotransmitter release. The inhibitory effect of trichloroethane on calcium currents may be involved in trichloroethane-induced sensory discomfort.     

HMJ-53A accelerates slow inactivation gating of voltage-gated K+ channels in mouse neuroblastoma N2A cells

Voltage-gated K⁺ (Kv) channels are important in repolarization of excitable cells such as neurons and endocrine cells. Kv channel gating exhibits slow inactivation (slow current decay) during continuous depolarization. The molecular mechanism involved in such slow inactivation is not completely understood, but evidence has suggested that it involves a restriction of the outer channel pore surrounding the selectivity filter. Pharmacological tools probing this slow inactivation process are scarce. In this work we reported that bath application of HMJ-53A (30 μM), a novel compound, could drastically speed up the slow decay (decay τ = 1677 ± 120 ms and 85.6 ± 7.7 ms, respectively, in the absence and presence of HMJ-53A) of Kv currents in neuroblastoma N2A cells. HMJ-53A also significantly left-shifted the steady-state inactivation curve by 12 mV. HMJ-53A, however, did not affect voltage-dependence of activation and the kinetics of channel activation. Intracellular application of this drug through patch pipette dialysis was ineffective at all in accelerating the slow current decay, suggesting that HMJ-53A acted extracellularly. Blockade of currents by HMJ-53A did not require an open state of channels. In addition, the inactivation time constants and percentage block of Kv currents in the presence of HMJ-53A were independent of the (i) degree of depolarization and (ii) intracellular K⁺ concentration. Therefore, this drug did not appear to directly occlude the outer channel pore during stimulation (depolarization). Taken together, our results suggest that HMJ-53A selectively affected (accelerated) the slow inactivation gating process of Kv channels, and could thus be a selective and novel probe for the inactivation gate.     

Depolarization evoked co-release of tachykinins from enteric nerves in the guinea-pig proximal colon

The aim of this study was to assess at which extent an even co-release of the tachykinins, substance P (SP) and neurokinin A (NKA), occurs from enteric neurons/nerves of the guinea-pig proximal colon during graded depolarization. In this preparation, a sharply diverging NK₁/NK₂ receptor pattern of tachykininergic co-transmission has been observed in physiological studies. The experiments were performed in capsaicin-pretreated (10μM for 15min) mucosa-free smooth muscle of guinea-pig proximal colon, to exclude the mucosa and the peripheral endings of primary afferent nerves as possible sources of released tachykinins. The content of extractable tachykinins was measured as SP- and NKA-like immunoreactivities (-LI) by radioimmunoassay. Chromatographic characterization of aqueous acetic acid extracts showed one peak of SP-LI corresponding to authentic SP, whereas there were multiple peaks of NKA-LI, the major one co-eluting with authentic NKA. An increased outflow of both SP- and NKA-LI was evenly produced in a concentration-dependent manner when the preparations were superfused with a high potassium (K) medium in which NaCl had been replaced with equimolar amounts (20–100mM) of KCl. The high K-evoked release of SP- and NKA-LI was dependent upon the presence of extracellular calcium and was inhibited by about 50% in the presence of the N-type voltage-dependent calcium channel blocker, ω-conotoxin GVIA (0.1μM). ω-Conotoxin MVIIC (1μM), a non-selective blocker of N-, P- and Q-type voltage-dependent calcium channels, likewise produced about 40% inhibition of evoked release of both peptides. No evidence for a role of L-type channels in tachykinin release was obtained, since the addition of nifedipine (1μM) or Bay K8644 (1μM) did not significantly affect the response to high K. Neither NK₁ receptor agonist (septide, 0.1μM) or antagonist(SR 140333, 10nM) nor NK₂ receptor agonists ([βAla⁸]NKA(4-10) and GR 64349, 0.1μM each) or antagonist (SR 48968, 10nM) did affect the high K-evoked release of tachykinins. We conclude that SP and NKA are evenly co-released in response to graded depolarization of enteric nerves in the guinea-pig colon. Therefore, the specialization of tachykininergic transmission observed in functional studies does not originate at the prejunctional level. The co-release of tachykinins involves the influx of extracellular calcium via N-type but not L-type calcium channels. No evidence for the presence of NK₁ or NK₂ autoreceptors affecting tachykinin release from enteric neurons was obtained. Received: 29 September 1997 / Accepted: 1 December 1997     

Functional impact of the hyperpolarization‐activated current on the excitability of myelinated A‐type vagal afferent neurons in the rat

1. The hyperpolarization‐induced, cation‐selective current I_h is widely observed in peripheral sensory neurons of the vagal and dorsal root ganglia, but the peak magnitude and voltage‐ and time‐dependent properties of this current vary widely across afferent fibre type. 2. Using patch clamp investigations of rat isolated vagal ganglion neurons (VGN) identified as myelinated A‐type afferents, we established a compendium of functional correlates between changes in membrane potential and the dynamic discharge properties of these sensory neurons as a result of the controlled recruitment of I_h using the current clamp technique. 3. Two robust reponses were observed in response to hyperpolarizing step currents: (i) upon initiation of the negative step current, there was a rapid hyperpolarization of membrane potential followed by a depolarizing voltage sag (DVS) towards a plateau in membrane potential as a result of steady state recruitment of I_h; and (ii) upon termination of the negative step current, there was a rapid return to the pretest resting membrane potential that often led to spontaneous action potential discharge. These data were strongly correlated (r² > 0.9) with a broad compendium of dynamic discharge characteristics in these A‐type VGN. 4. In response to depolarizing step currents of increasing magnitude, the discharge frequency of the A‐type VGN responded with increases in the rate of sustained repetitive discharge. Upon termination of the depolarizing step current, there was a post‐excitatory membrane hyperpolarization of a magnitude that was strongly correlated with action potential discharge rate (r² > 0.9). 5. Application of the selective hyperpolarization‐activated cyclic nucleotide gated (HCN) channel blockers ZD7288 (10 μmol/L) or CsCl (1.0 mmol/L) abolished I_h and all of the aforementioned functional correlates. In addition to reducing the excitability of the A‐type VGN to step depolarizing currents. 6. Because there is increasing evidence that the HCN channel current may represent a valid target for pharmacological intervention, the quantitative relationships described in the present study could potentially help guide the molecular and/or chemical modification of HCN channel gating properties to effect a particular outcome in VGN discharge properties, ideally well beyond merely selective blockade of a particular HCN channel subtype.     

Imidazole antimycotics affect the activity of various ion channels and insulin secretion in mouse pancreatic B-cells

In the present paper the effects of antimycotics with imidazole structure on the activity of various ion currents of mouse pancreatic B-cells and insulin secretion from isolated islets have been studied. Clotrimazole (0.1– 10 μM, bath solution without albumin) reversibly inhibited the whole-cell K⁺ _ATP current studied with the patch-clamp technique and concomitantly depolarized the membrane potential. Two other structurally related compounds, econazole and ketoconazole, exhibited similar effects on the whole-cell K⁺ _ATP current. Clotrimazole also inhibited the current through single K⁺ _ATP channels measured in the inside-out configuration. According to these results it seems unlikely that a cytoplasmic factor is involved in the action of clotrimazole on K⁺ _ATP currents. Clotrimazole (10 μM) also reduced the current through voltage-dependent Ca²⁺ and K⁺ channels and altered inactivation kinetics. Moreover, clotrimazole reversibly abolished a recently described inward current which is induced by hypotonic cell swelling. The results show that clotrimazole altered the activity of all ion currents in B-cells investigated in this study. Clotrimazole (3–100 μM, solution with albumin) irreversibly inhibited insulin secretion from isolated islets. With econazole and ketoconazole similar effects on hormone release were observed. The changes in the activity and kinetics of voltage-dependent Ca²⁺ and K⁺ currents are likely to contribute to the observed inhibition of insulin secretion. However, we cannot entirely rule out that imidazole antimycotics also interfere with a step in stimulus-secretion coupling distal to changes in membrane potential. Received: 9 July 1997 / Accepted: 29 July 1997     

Role of primary sensory neurons in the central effects of nicotine

Utilizing single unit recording techniques the nicotine-induced excitation of noradrenaline (NA)-containing neurons in the locus coeruleus (LC) was analyzed. Low doses of nicotine (40–160 g, IV) were found to dose-dependently increase the LC firing rate. The effect was antagonized by pretreatment with the quaternary ganglionic blockers hexamethonium (12 mg/kg, IP) and chlorisondamine (0.3 mg/kg, IV). Also, neonatal treatment with capsaicin, a procedure that is associated with a selective degeneration of primary sensory C-fibre afferents, clearly antagonized the effect of nicotine on LC neurons. The typical effect of nicotine on LC discharge was, in all essentials, mimicked by the quaternary nicotinic agonist tetramethylammonium (TMA). We here propose that the action of nicotine on central NA neurons is primarily executed peripherally via activation of primary sensory C-fibre afferents.     

Amitriptyline modulation of Na(+) channels in rat dorsal root ganglion neurons

The effects of amitriptyline, a tricyclic antidepressant, on tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) currents in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp method. Amitriptyline blocked both types of Na(+)currents in a dose-and holding potential-dependent manner. At the holding potential of -80 mV, the apparent dissociation constants (K(d)) for amitriptyline to block tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) channels were 4.7 and 105 microM, respectively. These values increased to 181 and 193 microM, respectively, when the membrane was held at a potential negative enough to remove the steady-state inactivation. Amitriptyline dose-dependently shifted the steady-state inactivation curves in the hyperpolarizing direction and increased the values of the slope factors for both types of Na(+) channels. The voltage dependence of the activation of both types of Na(+) channels was shifted in the depolarizing direction. It was concluded that amitriptyline blocked the two types of Na(+) channels in rat sensory neurons by modulating the activation and the inactivation kinetics.     

P2-Receptor-mediated inhibition of noradrenaline release in the rat pancreas

The aim of the study was to find out whether, and if so through which receptors, nucleotides modulate the release of noradrenaline in the rat pancreas. Segments of the pancreas were preincubated with [³H]-noradrenaline, superfused with medium containing desipramine (1μM) and yohimbine (1μM), and stimulated electrically, in most experiments by 60 pulses/1Hz. The adenosine A₁-receptor agonist N⁶-cyclopentyl-adenosine (CPA; EC₅₀ 32nM), the non-subtype-selective adenosine receptor agonists adenosine (EC₅₀ 15μM) and 5’-N-ethylcarboxamidoadenosine (NECA; EC₅₀ 135nM), and the nucleotides ATP (EC₅₀ 13μM), adenosine-5’-O-(3-thiotriphosphate) (ATPγS; EC₅₀ 19μM) and adenosine-5’-O-(2-thiodiphosphate) (ADPβS; EC₅₀ 16μM) decreased the evoked overflow of tritium. The adenosine A_2A-agonist 2-p-(2-carboxyethyl)-phenethylamino-5’-N-ethylcarboxamido-adenosine (CGS 21680) caused no change. The concentration-response curve of CPA was shifted to the right by the A₁-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 10nM; pK_d 9.1) but, like the concentration-response curve of adenosine, hardly affected by the P2-receptor antagonist cibacron blue 3GA (30μM). Combined administration of a high concentration of DPCPX (1μM) and 8-phenyltheophylline (10μM) abolished the effects of CPA and NECA. The concentration-response curves of ATP and ADPβS were shifted to the right by both DPCPX (10nM; pK_d 8.7 and 8.9, respectively) and cibacron blue 3GA (30μM; pK_d 5.0 and 5.2, respectively). The antagonist effects of DPCPX (10nM) and cibacron blue 3GA (30μM) against ATP were additive in a manner compatible with the blockade of two separate receptors for ATP. In the presence of the high concentration of DPCPX (1μM) and 8-phenyltheophylline (10μM), ATP and ADPβS still decreased evoked tritium overflow, and this decrease was attenuated by additional administration of cibacron blue 3GA (30μM). The P2-antagonists cibacron blue 3GA, reactive blue 2, reactive red 2, and to a limited extent also suramin and 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonate (XAMR0721), increased the evoked overflow of tritium by up to 114%. Pyridoxalphosphate-6-azophenyl-2’,4’-disulphonate (PPADS) caused no change. The results indicate that the postganglionic sympathetic axons of the rat pancreas possess A₁-adenosine and P2-receptors. Both receptors mediate an inhibition of noradrenaline release. The presynaptic P2-receptors are activated by an endogenous ligand, presumably ATP, during appropriate trains of action potentials. This is the first demonstration of presynaptic P2-receptors at postganglionic sympathetic neurons that are located in prevertebral ganglia. Received: 6 November 1997 / Accepted: 6 January 1998     

Inhibition of frog skeletal muscle sodium channels by newly synthesized chiral derivatives of mexiletine and tocainide

To search for potent use-dependent blockers of skeletal muscle sodium channels as potential antimyotonic agents, the actions of newly synthesized chiral analogs of mexiletine and tocainide were tested in vitro on sodium currents of single fibers of frog semitendinosus muscle by vaseline-gap voltage clamp method. The effect of each drug on the maximal peak Na⁺ transient (I_{Na max}) was evaluated as both tonic and use-dependent block by using infrequent depolarizing stimulation and trains of pulses at 2–10 Hz frequency, respectively. The mexiletine analog 3-(2,6-dimethylphenoxy)-2-methylpropanamine (Me2), having an increased distance between the phenyl and the amino groups, was less potent than mexiletine in producing a tonic block but produced a remarkable use-dependent block. In fact, the half-maximal concentration (IC₅₀) for tonic block of S(–)-Me2 was 108 μM vs. 54.5 μM of R(–)-mexiletine, but the IC₅₀ was 6.2 times lowered by the 10 Hz stimulation with respect to the 2.4fold decrease observed with mexiletine. The R(–)-mexiletine and the S(–)-Me2 were about twofold more potent than the corresponding enantiomers in producing a tonic block, but the stereoselectivity attenuated during use-dependent blockade. The more lipophilic 2-(4-chloro-2-methylphenoxy)-1-phenylethylamine (Me1), presently available as raceme, produced a potent and irreversible tonic block of the sodium currents with an IC₅₀ of 29 μM, but had a less pronounced use-dependent inhibition, with a 1.9fold decrease of the IC₅₀ at 10 Hz. The R(–) isomer of 2′,6′-valinoxylidide (To1), a tocainide derivative with an increased hindrance on the chiral carbon atom, was twofold (IC₅₀ = 209 μM) and tenfold (IC₅₀ = 27.4 μM) more potent than R(–)-tocainide in tonic and use-dependent block, respectively. Tocainide was almost devoid of stereoselectivity, whereas the eudismic ratio of To1 [(IC₅₀ S(+)-To1/IC₅₀ R(–)-To1] was 1.7. As for mexiletine and Me2, the stereoselectivity of To1 was the weaker the higher the frequency of stimulation. The cyclic pyrrolo-imidazolonic tocainide analog To2 produced a small tonic block at 500 μM, and 1 min stimulation at 10 Hz was needed to show up a 50% block of I_{Na max}. All the compounds produced a left-shift of the steady-state inactivation curve correlated positively with the extent of use-dependent inhibition, with the exception of the cyclic To2 that acted as an open-channel blocker. The highly use-dependent blockers Me2 and To1 might be promising drugs to solve high frequency discharges of action potentials typical of myotonic muscles. Concomitantly the high potency of Me1 and the open-channel block exerted by To2 can represent important features to get selective blockers for skeletal muscle sodium channels. Received: 12 March 1997 / Accepted: 7 August 1997     

Subtype determination of soma-dendritic α2-autoreceptors in slices of rat locus coeruleus

The aim of the study was to subclassify the soma-dendritic α₂-autoreceptors in the locus coeruleus (LC) of the rat by means of antagonists. To this end, the frequency of spontaneous action potentials was recorded extracellularly from single LC neurones in brain slices. The neurones fired spontaneously at an average rate of 1 Hz. The selective α₂-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) and noradrenaline decreased the action potential discharge with IC₅₀ values of 5 and 510 nM, respectively. The concentration-inhibition curves of UK 14,304 and noradrenaline were shifted to the right by phentolamine (0.15 μM) and rauwolscine (0.15 μM) but not by prazosin (1 μM). Apparent K _d values of phentolamine were 17 nM (against UK 14,304) and 20 nM (against noradrenaline). Apparent K _d values of rauwolscine were 47 nM (against UK 14,304) and 70 nM (against noradrenaline). (+)-Oxaprotiline (1 μM) suppressed the firing of the neurones within 10 to 33 min. In the continued presence of oxaprotiline, phentolamine and rauwolscine restored firing with EC₅₀ values of 120 and 250 nM, respectively. Prazosin (1 μM) again was ineffective. All three antagonist affinity estimates – against UK 14,304, exogenous noradrenaline and endogenous noradrenaline (that accumulates in the extracellular space in the presence of oxaprotiline) – yield an affinity order phentolamine > rauwolscine >> prazosin, prazosin being ineffective even at a concentration of 1 μM. These findings identify the soma-dendritic α₂-autoreceptors of the LC as the rat variant of the α_2A/D-adrenoceptor, i.e. α_2D. Not only presynaptic but also soma-dendritic α₂-autoreceptors may at least predominantly be α_2A/D throughout the nervous system. Received: 3 March 1997 / Accepted: 21 April 1997     

P2-receptor antagonists: IV. Blockade of P2-receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2

Effects of reactive blue 2 and twelve structurally related compounds were studied on contractions of the rat vas deferens elicited by α,β-methylene ATP (α,β-MeATP; mediated by P2X-receptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5′-O-(2-thiodiphosphate) (ADPβS; mediated by P2Y-receptors), and the degradation of ATP by rat vas deferens tissue. All compounds, except acid blue 41 and acid blue 129 (at up to 100μM), shifted the concentration-response curve of α,β-MeATP in the rat vas deferens to the right. Most increased, but uniblue A greatly decreased, the maximum of the curve. In the case of cibacron blue 3GA and reactive blue 19, of which three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from unity. The apparent K_d values of the effective substances ranged between 0.7 and 111μM. Most compounds increased the contraction of the rat vas deferens elicited by high K⁺. In the guinea-pig taenia coli, all compounds, except uniblue A and reactive blue 19 (at up to 100μM), shifted the concentration-response curve of ADPβS to the right in a parallel manner. In the case of acid blue 129 and acid blue 80, of which three concentrations were tested, the slope of the Arunlakshana-Schild regression did not differ from unity. The apparent K_d values of the effective substances were between 0.7 and 69μM. Most compounds also reduced the relaxation of the guinea-pig taenia coli elicited by noradrenaline. The removal of ATP from the medium by vas deferens tissue was decreased only by reactive blue 2, cibacron blue 3GA, uniblue A and reactive blue 19, with IC_25% values between 17 and 62μM. The structure-activity relationships for P2X- and P2Y-receptor blockade in this series are strikingly dissimilar. In reactive blue 2 and its isomers, for example, both the 1-amino-anthraquinone-2-sulphonate core and the ‘side-chain’ of the molecule are involved in P2X-receptor binding; P2Y-receptor affinity, in contrast, resides largely or totally in the anthraquinone core. The most promising antagonists are uniblue A which is P2X- versus P2Y-selective and acid blue 129 which is P2Y- versus P2X-selective, both with few, if any, non-P2-receptor effects at concentrations blocking the respective P2-subtype. Received: 23 July 1997 / Accepted: 4 November 1997     

Idebenone protects hippocampal neurons against amyloid β-peptide-induced neurotoxicity in rat primary cultures

The application of amyloid β-peptide (Aβ) 1–40 (10 μM) caused neurodegeneration of hippocampal neuronal cells, as indicated by the release of lactate dehydrogenase (LDH) into the culture medium. Treatment with idebenone (10–1000 nM), a potent antioxidant in mitochondria, protected the hippocampal neurons against the Aβ1–40 (10 μM)-induced neurotoxicity. To determine the morphological change in neurons during the Aβ1–40-induced cytotoxicity, the cells were immunostained with anti-MAP2 antibodies. After 4-day exposure to 10 μM Aβ1–40, the number of neurons was reduced, and the surviving neurons had an apparently reduced number of neurites which were shorter than those of control neurons. When idebenone was added to the culture medium with Aβ1–40, the number of surviving neurons was significantly increased, and their neurites were as long as seen in control culture. These results suggest that reactive oxygen species mediate neurotoxicity of Aβ1–40, and idebenone protects neurons against the Aβ1–40-induced neurotoxicity. Received: 5 June 1998 / Accepted: 25 August 1998     

低浓度美蓝染色在乳头溢液手术中的应用

目的探讨乳管内低浓度美蓝染色并回抽的方法在乳头溢液患者手术中的作用。方法 43例乳头溢液行手术治疗的患者,随机分为改良组(低浓度美蓝染色并回抽)25例,传统手术组(乳管内高浓度美蓝染色)18例,分析两组术中美蓝外溢污染手术野以及微小病变的识别情况。结果 43例乳头溢液的病变导管均染色成功,改良组美蓝外溢污染手术野4例(16%),传统组8例(44.4%);改良法比传统方法更容易识别乳管微小病变。结论改良法对乳头溢液患者有应用价值。