Petrus J. Pauwels

Ca++ and Na+ channels involved in neuronal cell death. Protection by flunarizine

Flunarizine, a class IV Ca++ antagonist non-selective for slow Ca++ channels, has been shown to be beneficial in the prophylactic treatment of migraine, the treatment of vertigo, and as add-on treatment in therapy-resistant forms of epilepsy. Flunarizine protects the brain against functional and/or structural neuronal damage in various animal models of cerebral ischemia. In addition to its cerebrovascular effect, flunarizine has also direct neuroprotective actions. New data have emerged on flunarizine with regard to Ca++ and Na+ channels in neuronal cells. There are several possible mechanisms involved in the mode of action of flunarizine. Flunarizine may block Ca++ and Na+ channels, both of which may flux Ca++ as well as Na+. A decrease in Ca++ influx may prevent further release of glutamate, and activation of NMDA receptor gated Ca++ channels at physiological pH. A decrease in Na+ influx may prevent cytotoxicity secondary to a large gain in intracellular Ca++, by reverse operation of the Na+/Ca++ exchanger. This mechanism may be important when the glycolytic rate is increased with concomitant acidosis, and phospholipids are broken down as occurs typically during ischemia. Given the complexity of biochemical events leading to cell death, blocking exclusively one channel subtype is not likely to yield sufficient protection. Hence, it may be useful to develop anti-ischemic compounds which act on a series of pathways involved in Ca++ overload, rather than selectively block one such channel.

Blockade of nitric oxide formation does not prevent glutamate-induced neurotoxicity in neuronal cultures from rat hippocampus

This study examined the role of nitric oxide (NO) in glutamate-induced, N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in rat hippocampal neuronal cultures grown under serum-free conditions. Formation of cGMP was used as an indirect measure of NO formation. Neuronal cell degeneration was monitored by measuring the release of lactate dehydrogenase (LDH). Neuronal cells showed a 4-fold increase in cGMP formation and release of LDH upon exposure to 30 microM glutamate. cGMP formation was fully inhibited by 1 microM nitro-arginine (N-Arg), 100 microM hemoglobin or 1 microM MK-801. In the presence of 1 microM MK-801, glutamate induced neither cGMP formation nor neuronal cell degeneration. However, when NO formation was inhibited by means of 100 microM N-Arg, glutamate still induced neurotoxicity. Therefore, in serum-free hippocampal cultures glutamate neurotoxicity occurs notwithstanding complete inhibition of the NO-synthase enzyme by N-Arg. Our data provide evidence that NO, synthesized upon glutamate exposure, has not a primary toxic action in pure hippocampal neuronal cultures.

Activity of serotonin (5-HT) receptor agonists, partial agonists and antagonists at cloned human 5-HT1a receptors that are negatively coupled to adenylate cyclase in permanently transfected hela cells

The activity of serotonin (5-HT) receptor agonists, partial agonists and antagonists, and various other neurotransmitter receptor antagonists at human 5-HT1A receptors that are negatively coupled to adenylate cyclase in permanently transfected HeLa cells was investigated. 5-HT1A receptor-mediated inhibition of adenylate cyclase was studied by measuring inhibition of cAMP accumulation, induced by forskolin. At 100 microM forskolin produced a 100-fold increase in cAMP formation: 5-HT concentration dependently inhibited the cAMP formation; maximal inhibition was attained at 1 microM 5-HT and represented 90% of the stimulated cAMP formation. Full inhibition was observed with 5-HT1A receptor agonists: N,N-dipropyl-8-hydroxy-2-aminotetralin (8-OH-DPAT) and flesinoxan, and non-selective 5-HT receptor agonists: d-lysergic acid diethylamide (d-LSD), RU 24,969, bufotenine, methysergide and tryptamine. The rank order of potency of the compounds for inhibiting the cAMP formation corresponded to the rank order of the binding affinities of the drugs for the 5-HT1A receptor. Partial inhibition was obtained with submicromolar concentrations of buspirone, spiroxatrine and ipsapirone. A slight inhibition was observed with 1 microM 5-HT receptor agonist CP 93129 and 1 microM 5-HT receptor antagonists mesulergine and BW-501. No inhibition was found with: the 5-HT receptor agonists quipazine, sumatriptan and 1-(2,5-dimethoxy-4-methylphenyl)-2- aminopropane (DOM); the 5-HT receptor antagonist ICS-205,930; and other neurotransmitter receptor antagonists such as pindolol, CGP 20712-A, prazosin, sulpiride and pyrilamine. Spiperone and pindolol fully antagonized the agonist-mediated inhibition of forskolin-stimulated cAMP formation. Partial inhibition of the agonist-mediated inhibition of forskolin-stimulated cAMP formation was apparent with 1 microM ocaperidone and 1 microM ipsapirone. It can be concluded that HeLa cells, permanently expressing human 5-HT1A receptors, are a valid cellular system for studying the negative coupling of 5-HT1A receptors to adenylate cyclase and the action of compounds thereupon.

Stimulated [35S]GTP gamma S binding by 5-HT1A receptor agonists in recombinant cell lines. Modulation of apparent efficacy by G-protein activation state.

Abstract G-protein activation by different 5-HT receptor ligands was investigated in h5-HT1A receptor-transfected C6-glial and HeLa cells using agonist-stimulated [35S]GTPγS binding to membranes in the presence of excess GDP. 5-HT (10 μM) stimulated [35S]GTPγS binding in the C6-glial membrane preparation to a larger extent than in the HeLa preparation; maximal responses with 30 μM GDP were 490 ± 99 and 68 ± 12%, respectively. With the 5-HT receptor agonists that were being investigated, the two preparations displayed the same rank order of potency for stimulation of [35S]GTPγS binding. In the C6-glial preparation at 0.3 μM GDP, the rank order of maximal effects was: 5-HT (1.00) > 8-OH-DPAT (0.90) = R(+)-8-OH-DPAT (0.87) = 5-CT (0.86) = L694247 (0.84) > S(–)8-OH-DPAT (0.68) = buspirone (0.67) = spiroxatrine (0.67) = flesinoxan (0.64) > ipsapirone (0.53) = (–)-pindolol (0.50) > SDZ216525 (0.25). However, differences in maximal response in the C6-glial preparation were magnified by increasing the GDP concentrations, indicating that the activity state of G-proteins can affect the maximal response. With the exception of 5-CT and L694247, increasing the amount of GDP to 30 μM and higher concentrations resulted in an attenuation of both the ligand’s maximal effect (24 to 56%) and apparent potency (6 to 24-fold). Each of the [35S]GTPγS binding responses was mediated by a 5-HT1A receptor as indicated by the competitive blockade by WAY100635 and spiperone. Only 5-CT and L694247 in some conditions displayed an efficacy similar to that of 5-HT at the h5-HT1A receptor; the other agents with intrinsic activity are partial agonists at this receptor. The data also suggest that the activity state of the G-proteins is involved in the maximal effects that can be produced by activating the h5-HT1A receptor.

[3H]Batrachotoxinin a 20-α-benzoate binding to sodium channels in rat brain: Characterization and pharmacological significance

Attempts were made to find whether [3H]batrachotoxinin A 20-alpha-benzoate provides a specific probe for measuring interactions of local anesthetics with the sodium channel complex. [3H]Batrachotoxinin A 20-alpha-benzoate binding, [14C]guanidine and 22Na uptake were investigated in rat brain crude synaptosomal preparations and the potencies of drugs belonging to various chemical and pharmacological classes were compared. The results show that [14C]guanidine uptake seems to be a good model for measuring Na+ fluxes. An allosteric interaction between site 2 and 3 of the Na+ channel is apparent since (i) scorpion venom was able to increase further the guanidine or Na+ stimulation of site 2 toxins to a maximal level and (ii) in the presence of scorpion venom, binding of [3H]BTX-B to site 2 of the Na+ channel was enhanced. A good correlation exists between drug potencies in the binding and uptake experiments. Inhibition by drugs was not restricted to drugs known as local anesthetics. It can be concluded that many drugs seem to interfere with the Na+ channel of rat brain. This may play a role not only in the major effects of local anesthetic drugs but also in the side-effects of drugs from other classes.

Flunarizine inhibits a high-threshold inactivating calcium channel (N-type) in isolated hippocampal neurons

The action of flunarizine on the high-threshold inactivating calcium channel (N-type) in hippocampal neurons of the rat was investigated using the whole-cell voltage clamp technique. Flunarizine reduced the currents at all test potentials, without shifting the peak of the current-voltage relationship along the voltage-axis. The drug did not affect the activation curve, but drastically decreased the slope conductance in the linear region of the current-voltage relationship. Block of the current by flunarizine occurred in a use-dependent way. Flunarizine was without effect when applied intracellulary, and the onset of action, when applied extracellularly, was slow (range of minutes). The Kd for the block by flunarizine obtained after 6 repetitive depolarizations at 0.2 Hz (pulse duration 150 ms) from -90 mV to 0 mV was 0.8 microM. In conclusion, we present electrophysiological evidence that flunarizine blocks the high-threshold inactivating Ca channel of hippocampal neurons of the rat. We discuss the possibility that flunarizine might inhibit neuronal transmitter release by means of this effect.

Isolated rat cardiac myocytes as an experimental model to study calcium overload: the effect of calcium-entry blockers

Calcium overload and the effect of a series of calcium-entry blockers were studied in isolated adult cardiac myocytes from the rat challenged with veratrine. The isolation procedure resulted in a high yield of individual rod shaped, calcium tolerant myocytes. After incubation with veratrine, an alkaloid which induces both sodium and calcium influx, 93% of the myocytes became calcium intolerant: the quiescent rod shaped cells vigorously contracted after 30 sec of contact with veratrine and contracture (round cells) ensued within 1 min. Exposure for 30 min to various doses of calcium-entry blockers prior to veratrine addition resulted in the prevention of contracture, the degree of protection depending on the type and the concentration of calcium-entry blocker. Among the different calcium-entry blockers tested, the diarylalkylpiperazines lidoflazine, cinnarizine and flunarizine were protective from the 10(-7) M concentration onwards. Nicardipine was protective at the 10(-6) M and 10(-5) M concentrations, verapamil at 10(-5)M only while other blockers of the slow channel type (diltiazem and nifedipine) were not protective in the concentration range tested. This study shows that isolated myocytes represent a valid model for pharmacological investigations. The results with the calcium-entry blockers stress the heterogeneity of the different series of calcium-entry blockers.

Comparison of in vitro binding properties of a series of dopamine antagonists and agonists for cloned human dopamine D2S and D2L receptors and for D2 receptors in rat striatal and mesolimbic tissues, using [125I] 2'-iodospiperone.

We investigated the ligand binding properties in vitro of two splice variants of the cloned human dopamine D2 receptor (the 443 and 414 amino acids long forms called D2L and D2S, respectively), expressed in 293 human kidney cells, in comparison with those of the dopamine D2 receptors in rat striatum, nucleus accumbens and tuberculum olfactorium. The new radioligand, [125I]2′-iodospiperone, showed a similar high binding affinity (KD:0.056–0.122 nM) for cloned human D2S and D2L receptors and for the D2 receptors in the three rat brain areas. Binding affinities of 25 dopamine antagonists and of 10 dopamine agonists belonging to different chemical classes were measured. The IC50 values of the antagonists were virtually identical in the five preparations: spiperone was the most potent compound (pIC50 ≲ 9.9), remoxipride the least potent one (pIC50 ≲ 5.7). The agonists showed similar IC50 values for the cloned human D2S and D2L receptors but their affinity for rat brain D2 receptors was 2- to 5-fold higher. Dopamine showed shallow inhibition curves, the high affinity binding was 10-fold lower for the cloned human D2 receptors than for the rat brain D2 receptors. Addition of stable guanosine-5′-triphosphate (GTP) analogues shifted the D2 receptors in the rat brain tissues to the “low” affinity state, the low affinity binding of dopamine was equal to the affinity for the cloned human receptor. None of the dopamine antagonists or agonists could differentiate between the two splice forms of the cloned human D2 receptors or between the D2 receptors in rat striatal and mesolimbic tissues. The lower apparent affinity of some agonists and of dopamine in the absence of stable GTP analogues suggests a less appropriate receptor G-protein coupling for the cloned human D2 receptors expressed in the 293 human kidney cells. Unexpectedly, guanosine-5′-O-(3-thiotriphosphate) (GTP-γ-S) reduced the [125I]2′-iodospiperone binding to the D2 receptors by 20–35% in the rat brain tissues and the cloned human D2L receptor, and by 75% to the cloned human D2S receptor. The inhibition in the last case could be prevented partly by submicromolar concentrations of dopamine. The GTP-γ-S effect is suggested to be due to reduction of disulphide bonds in the receptor. Recent molecular modelling studies indicated an important role of the disulphide bridge between Cys107 at the start of transmembrane domain three and Cys182 in the third extracellular loop, for the binding of dopamine to the D2 receptor.

Rapid desensitization and resensitization of 5-HT2 receptor mediated phosphatidyl inositol hydrolysis by serotonin agonists in quiescent calf aortic smooth muscle cells

Agonist regulation of 5-hydroxytryptamine2 (5-HT2) receptors was studied in calf aortic smooth muscle cultures incubated in a quiescent, defined synthetic medium that does not stimulate cell proliferation, but that provides cells with supplements that maintain cell viability. In these cells, 5-hydroxytryptamine (5-HT)-induced [3H]inositol phosphates accumulation showed the characteristics of a 5-HT2 receptor coupled transducing system according to the inhibition of the response by 5-HT2 antagonists at nanomolar concentrations. The 5-HT2 receptor coupled response became rapidly desensitized during continued incubation with 5-HT and 1-(2,5-dimethoxy-4-methylphenyl)-2- aminopropane (DOM); nearly full desensitization was obtained in two hours with 10 microM 5-HT and DOM pretreatment. The recovery of the response had a half-live of 5 hours after 2 hours pretreatment and of 9.5 to 12.5 hours after 24 to 96 hours agonist pretreatment. The DOM-induced desensitization of the 5-HT2 receptor coupled response was fully blocked by 0.1 microM cinanserin. Cinanserin alone did not induce desensitization or up-regulation of the 5-HT2 receptor coupled response at 0.1 microM. It may be that the down-regulation of central 5-HT2 receptors by antagonists in vivo is a heterologous process due to mediators which are triggered by 5-HT2 antagonistic action.

Attenuation of neurotoxicity following anoxia or glutamate receptor activation in EGF- and hippocampal extract-treated neuronal cultures

Neurotoxicity following anoxia or glutamate receptor activation was studied in primary neuronal cultures grown in serum-free, chemically defined CDM R12 medium. Exposure to 1 mM KCN, 0.5 mM kainic acid and 0.5 mM N-methyl-D-aspartate led to progressive neuronal degeneration. This damage was quantified by measuring lactate dehydrogenase released in the culture medium. The toxic effects were observed early during the development of the neuronal culture (from 4 days in vitro on) and seemed to be neuron-specific since astrocyte cultures were not affected. Chronic treatment of the neuronal cultures with epidermal growth factor at 10 ng/ml and hippocampal extract at dil. 1/833 (w/v) induced morphological alterations, increased beta-adrenergic receptor coupled adenylate cyclase activity, increased level of total lactate dehydrogenase activity in the case of epidermal growth factor-treated cultures, and attenuation of lactate dehydrogenase release following exposure to KCN or glutamate receptor agonists. The alterations observed are probably due to the proliferation and differentiation of glial cells in these treated cultures. This suggests that glial cells protect neurons in vitro from degeneration induced by anoxia or glutamate receptor activation.