The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.     

EFFECT OF CROSS-FOSTERING ON NEONATAL SODIUM BALANCE AND ADULT BLOOD PRESSURE IN THE SPONTANEOUSLY HYPERTENSIVE RAT

1. The aim of the present study was to compare electrolyte handling in naturally reared neonatal spontaneously hypertensive rats (SHR) with those reared by a Wistar-Kyoto (WKY) rat foster mother (denoted SHRX), as cross-fostering SHR pups to a WKY rat dam lowers adult blood pressure in the SHR. 2. The electrolyte content of WKY rat and SHR dams’ milk was determined and electrolyte intake and urinary excretion rates were calculated in both naturally reared and cross-fostered WKY rat and SHR pups. 3. The milk sodium concentration fell in both strains (WKY rat: 31.8 ± 2.0 to 15.2 ± 1.2 mmol/L; SHR 31.9 ± 2.5 to 18.2 ± 1.6 mmol/L; P < 0.001), as did potassium (P < 0.001), over lactation, but there were no differences between strains. Calcium and magnesium concentrations increased (P< 0.001), although SHR dam's milk contained less calcium (P < 0.001) than that of WKY rat dams during the third week of lactation. 4. Spontaneously hypertensive rat pups ingested less milk (P<0.05) than WKY rat pups; therefore, their cumulative sodium intake over postnatal days 4–15 was significantly lower than that of WKY rat pups (WKY rat vs SHR: 84.4 ± 3.6 vs 59.7 ± 2.6 μmol/g bodyweight, respectively; P < 0.05) and fostered SHRX pups (77.7 ± 7.0 μmol/g bodyweight; P < 0.05). Potassium and magnesium intakes were comparable between SHR, WKY rat and SHRX pups, but SHR pups ingested significantly less calcium than either WKY rat pups (136.1 ± 6.4 vs 200.1 ± 9.5p, mol/g bodyweight, respectively; P<0.05) or SHRX pups (200.0 ± 18.0 μmol/g bodyweight; P<0.05). 5. These data show that the neonatal SHR experiences a period of sodium deficiency during the developmental stage when cross-fostering is effective in lowering blood pressure. This is consistent with the reported up-regulation of the renin-angiotensin system observed in SHR at this time and may have a long-term influence on blood pressure.     

DIFFERENCES IN ENDOTHELIUM-DEPENDENT RELAXATION IN VARIOUS ARTERIES FROM WATANABE HERITABLE HYPERLIPIDAEMIC RABBITS WITH INCREASING AGE

1. Endothelium-dependent relaxation in response to acetylcholine (ACh) and the calcium ionophore A 23187 was examined in aorta, coronary, basilar and renal arteries isolated from Watanabe heritable hyperlipidaemic (WHHL) rabbits of 2, 6 and 12 months of age, with normolipidaemic heterozygous WHHL rabbits as controls. 2. In the rings of WHHL rabbit aortae and coronary arteries preconstricted with vasoconstrictors, endothelium-dependent relaxation in response to ACh was attenuated with age compared to the heterozygous WHHL rabbits. A significant negative correlation was found between the total cholesterol content and the relaxation response to ACh in the aortae or coronary arteries from 6 and 12 month old WHHL rabbits. 3. In the rings of basilar arteries, endothelium-dependent relaxations to ACh were not modified with age. Similarly, in the rings of renal arteries, the relaxation response to ACh was not changed with age, but in the 6 and 12 month preparations, after the age of 6 months, a contraction following the relaxation appeared at higher concentrations of ACh (10^?7 to 10^?6 mol/L). The contraction was endothelium-dependent and inhibited by indomethacin. 4. A 23187-induced endothelium-dependent relaxations were also markedly attenuated in the aorta and significantly in the coronary artery with age. 5. Endothelium-independent relaxation to sodium nitroprusside was not changed in all arteries from WHHL rabbits of different ages. 6. These findings indicate that in the aorta and coronary artery of the WHHL rabbit, the endothelium-dependent relaxation to ACh and A 23187 becomes impaired with increasing age (i.e., with the progression of cholesterol deposition in the arterial wall) but is preserved in the basilar and renal artery.     

Inhibition of GABA-gated chloride channel function by arachidonic acid

The effects of arachidonic acid and its metabolites on gamma-aminobutyric acid (GABAA) receptor function were determined in rat cerebral cortical synaptoneurosomes. Incubation of synaptoneurosomes with phospholipase A2 decreased muscimol-induced 36Cl- uptake. Arachidonic acid, the major unsaturated fatty acid released by phospholipase A2, also inhibited muscimol-induced 36Cl uptake. Similar inhibition was obtained with other unsaturated fatty acids (docosahexaenoic, oleic) but not with saturated fatty acids (stearic, palmitic). The effect of arachidonic acid on muscimol responses was inhibited by bovine serum albumin (BSA), and BSA enhanced muscimol responses directly, indicating the generation of endogenous arachidonic acid in the synaptoneurosome preparation. The generation of endogenous arachidonic acid was also indicated by the ability of 2 inhibitors of arachidonic acid metabolism, indomethacin and nordihydroguaiaretic acid (NDGA), to inhibit muscimol-induced 36Cl uptake. We conclude that arachidonic acid probably has both direct and indirect actions on muscimol responses since both enzyme inhibitors inhibited muscimol responses but did not prevent the effect of exogenously added arachidonic acid. In additional experiments, arachidonic acid metabolites generated by cyclooxygenase, prostaglandins D2, E2 and F2 alpha, each decreased muscimol responses; prostaglandins F2 alpha was the most potent inhibitor. Since the unsaturated fatty acids and their metabolites are most susceptible to peroxidation, a generating system of superoxide radicals was tested on muscimol responses. A combination of xanthine and xanthine oxidase inhibited muscimol-induced 36Cl uptake in a concentration-dependent manner. We propose that the inhibition of GABAA neurotransmission by arachidonic acid and its metabolites can lead to increased neuronal excitability. This mechanism may play an important role in the development of neuronal damage following seizures or cerebral ischemia.     

Flunarizine induces a transient loss of tyrosine hydroxylase immunoreactivity in nigrostriatal neurons

Neurotoxic effects of flunarizine (Fz), a selective calcium channel blocker, on the nigrostriatal dopamine system was investigated. Systemic injections of Fz to mice resulted in a transient loss of tyrosine hydroxylase (TH) immunoreactive nigrostriatal neurons without cell loss. TH immunoreactivity in these neurons was greatly reduced as rapidly as one day after drug administration (regardless of dosage used) and thereafter recovered in both dose- and time-dependent manners. Such a novel neurotoxic action of Fz may constitute a morphological substrate for reversible drug-induced parkinsonian signs described in recent clinical case reports.     

Interaction between thiol-modifying agents and P1075, a KATP channel opener, in rat isolated aorta

In vascular smooth muscle, openers of ATP-dependent potassium channels (K _ATP channels), such as P1075 (N-cyano-N’-(1,1-dimethylpropyl)-N’’-3-pyridylguanidine), produce relaxation. In this study we have investigated the effects of thiol-modifying agents on the binding of P1075 and on the ⁸⁶Rb⁺ efflux stimulating and vasorelaxant effects of the opener in rat aortic rings. The increase in ⁸⁶Rb⁺ efflux induced by P1075 was taken as a qualitative measure of K⁺ channel opening. The hydrophilic SH-group-oxidizing substance, thimerosal (1 to 100μM), abolished specific binding of [³H]-P1075 with an IC₅₀ value of 7.6±1.2μM; at 30μM, the half time for inhibition was 38min. Two other thiol-oxidizing agents, PMB (4-hydroxy-mercuribenzoic acid) and DTBNP (2,2’-dithio-bis(5-nitropyridine)), inhibited binding up to 86% and 44%, respectively. The disulphide bond reducing substance, DTT (1,4-dithiothreitol, 0.1 to 1mM), reduced [³H]-P1075 binding by up to 20% and partially reversed the inhibitory effect of thimerosal. In ⁸⁶Rb⁺ efflux experiments, thimerosal (3 to 100μM) concentration-dependently increased basal efflux but inhibited P1075-stimulated tracer efflux with an IC₅₀ value of 7±1μM. The inhibitory effect occurred with a half-time of approximately 8min and was essentially reversed by DTT. In rings precontracted with noradrenaline, thimerosal inhibited the vasorelaxant effect in a noncompetitive manner, shifting the concentration-relaxation curves to the right and reducing maximum relaxation.The data show that oxidation of thiol groups interferes with the binding of the K _ATP channel opener, P1075; concomitantly, the ⁸⁶Rb⁺ efflux stimulating and the vasorelaxant effects are inhibited. Reduction of disulphide bonds by DTT has only minor effects on the action of P1075. Collectively, the results suggest that intact thiol groups are essential for the functioning of the K_ATP channel in rat aorta. The different kinetics governing the inhibition of opener binding and of opener-stimulated ⁸⁶Rb⁺ efflux suggest that the SH-groups involved in the two processes differ in their accessibility to thimerosal and/or in their reactivity. Received: 7 April / Accepted: 9 July 1997     

Na+ currents near and away from endplates on human fast and slow twitch muscle fibers

Fast and slow twitch muscle fibers have distinct contractile properties. Here we determined that membrane excitability also varies with fiber type. Na⁺ currents (I_NA) were studied with the loose-patch voltage clamp technique on 29 histochemically classified human intercostal skeletal muscle fibers at the endplate border and <200 μm from the endplate (extrajunctional). Fast and slow twitch fibers showed slow inactivation of endplate border and extrajunctional I_NA and had increased I_NA at the endplate border compared to extrajunctional membrane. The voltage dependencies of I_NA were similar on the endplate border and extrajunctional membrane, which suggests thatboth regions have physiclogically similar channels. Fast twitch fibers had larger I_NA on the endplate border and extrajunctional membrane and manifest fast and slow inactivation of I_NA at more negative potentials than slow twitch fibers. For normal muscle, the differences between I_NA on fast and slow twitch fibers might: (1) enable fast twitch fibers to operate at high firing frequencies for brief periods; and (2) enable slow twitch fibers to operate at low firing frequencies for prolonged times. Disorders of skeletal membrane excitability, such as the periodic paralyses and myotonias, may impact fast and slow twitch fibers differently due to the distinctive Na⁺ channel properties of each fiber type. © 1993 John Wiley & Sons, Inc.     

Analgesic profile of the sodium salt of pemedolac

Pemedolac Na, 1-ethyl-1,3,4,9-tetrahydro-4-(phenylmethyl)-pyrano [3,4-b] indole-1-acetic acid sodium salt, exhibited equipotent analgesic effects after oral, iv, and im administration, suggesting that it is well absorbed. In mouse writhing models, the ED₅₀ values ranged from 0.3 mg (0.81 μmol)/kg (vs. acetylcholine) to 4.3 mg (11.6 μmol)/kg (vs. paraphenylbenzoquinone [PBQ]). In the rat Randall-Selitto model, the ED₅₀ o the compound was approximately 0.001 mg (2.7 nmol)/kg, with a flat dose response curve. The peak effects lasted for 7–9 h, 10–18 h, and 5 h following oral, im, and iv injections, respectively. Intracerebroventricular (i.c.v.) injections of pemedolac Na inhibited the PBQ-induced writing in mice with an ED₅₀ of 43.5 μg (0.12 μmol)/mouse, and this effect was not antagonized by naloxone. It was inactive in the hot plate and tail flick tests, demonstrating that pemedolac Na does not act via an opiate mechanism. These results indicate that pemedolac Na is a viable parenteral and oral analgesic, typified by high analgesic potency, a rapid onset and long duration of action, and an extremely wide safety index. © Wiley-Liss, Inc.     

Down-regulation of Na channel expression by A23187 in NlE-115 neuroblastoma cells

Growth of cultured NlE-115 neuroblastoma cells in 1 μM A23187 for 2 days to elevate internal Ca reduced both membrane Na current and the transient, but not steady state, component of outward K current. Na channel mRNA abundance was reduced by an average value of 45% without effect on Kv3.1. Increases in internal Ca may therefore control excitability by independent regulation of Na and K channel mRNA abundance in neurons.     

Development of an animal model for neuroleptic malignant syndrome: heat-exposed rabbits with haloperidol and atropine administration exhibit increased muscle activity, hyperthermia, and high serum creatine phosphokinase level

The neuroleptic malignant syndrome (NMS) is a life-threatening complication of neuroleptic treatment. To elucidate the pathogenesis of NMS, an animal model has been developed. Experimental rabbits treated with haloperidol (1 mg/kg) by intramuscular injection, were studied for the diagnostic symptoms of increased muscle rigidity, elevated body temperature, and high serum creatine phosphokinase (CPK) level. Administration of haloperidol (1 mg/kg) and atropine (0.4 mg/kg), and exposure to high ambient temperature (35°C) induced a significant increase in electromyographic activity with muscle rigidity similar to that observed in patients with NMS. Such rabbits also showed elevated body temperature and serum CPK value. In addition to the similarity of the signs and symptoms, all parameters measured (muscle rigidity, body temperature, and serum CPK level) were normalized by dantrolene treatment. The effectiveness of dantrolene in the experimental animal partially confirms the validity of this animal model for NMS. This experimental animal model for NMS may be useful to elucidate the pathogenesis of NMS.