H.W.G.M. Boddeke

Functional expression of CXCR3 in cultured mouse and human astrocytes and microglia

It has been established recently that inflammation of the CNS is accompanied by an expression of chemokines within the CNS. Several lines of evidence suggest that chemokines within the CNS initiate and orchestrate the infiltration of the inflamed brain by blood leukocytes. It is also known that endogenous cells of the CNS express functional chemokine receptors, raising the possibility that chemokines may be involved in intercellular signalling between brain cells during brain inflammation. It was shown recently that two chemokine ligands for CXCR3 are induced rapidly in damaged neurons. Little is known yet on the function of neuronal chemokine expression. In order to investigate whether neuronal chemokines contribute to endogenous signalling within the CNS we investigated possible expression of CXCR3 in glial cells. Reverse transcription-polymerase chain reaction experiments and in situ hybridization analysis showed that cultured astrocytes and microglia from both mouse and human sources express CXCR3 mRNA. Protein expression of CXCR3 in both cell types was detected by immunocytochemistry. Moreover, stimulation of cultured glial cells with chemokine ligands for CXCR3 induced intracellular calcium transients and chemotaxis, indicating the functional expression of CXCR3. These results indicate that glial cells in culture functionally express the chemokine receptor CXCR3. Since it has been shown that brain damage rapidly induces expression of neuronal chemokines that activate CXCR3, we suggest that glial CXCR3 might contribute to an intercellular signalling system in the CNS related to pathological conditions.

CENTRAL 5-HT1A RECEPTORS AND THE MECHANISM OF THE CENTRAL HYPOTENSIVE EFFECT OF (+)8-OH-DPAT, DP-5-CT, R28935, AND URAPIDIL

This study investigated the central hypotensive effects of drugs that possess a high affinity for central 5-hydroxytryptamine (5-HT1A) binding sites; (+)8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), N,N-dipropylcarboxamidotryptamine (DP-5-CT), erythro-1-(1-[2(1,4-benzodioxan-2-yl)-2-hydroxyethyl]- 4-piperidyl)-2-benzimidazolinone (R28935) and urapidil proved to possess high affinity and selectivity for central 5-HT1A binding sites, labeled by [3H]8-OH-DPAT. (+)8-OH-DPAT (0.1-10 micrograms/kg) given through the left vertebral artery of chloralose-anesthetized cats, lowered blood pressure by a biphasic dose-response curve. When given systemically, 10- to 100-fold higher doses of (+)8-OH-DPAT were necessary to obtain the same hypotensive effect when compared with central administration. Besides 8-OH-DPAT, R28935, DP-5-CT and urapidil also lowered blood pressure by a central mechanism in doses that were ineffective when given systemically. The central hypotensive effect of 0.3 micrograms/kg (+)8-OH-DPAT, 3 micrograms/kg DP-5 CT, and 3 micrograms/kg R28935 could be blocked by 100 micrograms/kg (-)pindolol, indicating that central 5-HT1A receptors are involved. High doses of (+)8-OH-DPAT (3-10 micrograms/kg) can also lower blood pressure by activating central alpha 2-adrenoceptors. The hypotensive effect of 300 micrograms/kg urapidil given through the vertebral artery could not be blocked by (-)pindolol. These results indicate the involvement of central 5-HT1A receptors in the mechanism of the central hypotensive activity of (+)8-OH-DPAT, DP-5-CT, and R28935.

Expression of the low affinity neurotrophin receptor p75 in spinal motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a lethal neurodegenerative disorder involving motoneuron loss in the cortex, brainstem and spinal cord, resulting in progressive paralysis. Aberrant neurotrophin signalling via the low affinity neurotrophin receptor p75 has been suggested to be involved in the motoneuron death by the activation of apoptotic pathways. In order to investigate the involvement of neurotrophin receptor p75 in the amyotrophic lateral sclerosis related motoneuron degeneration process, we have studied the expression of this receptor in the spinal cord of transgenic mice carrying a mutated human Cu, Zn superoxide dismutase gene. Mutations in the superoxide dismutase gene are one of the genetic causes for familiar amyotrophic lateras sclerosis and human superoxide dismutase-1 transgenic mice develop symptoms and pathology similar to those in human amyotrophic lateras sclerosis. Our study shows that in these mice, spinal motoneurons, which normally do not contain the neurotrophin receptor p75 receptor, express this receptor during the progress of the disease. Expression of the neurotrophin receptor p75 receptor coincides with the expression of activating transcription factor 3, a member of the activating transcription factor/cyclic AMP family of stress transcription factors. Only a minority of these spinal motoneurons actually showed co-expression of neurotrophin receptor p75 with caspase-3 activity, suggesting that expression of the neurotrophin receptor p75 receptor is not directly related to the execution phase of the apoptosis process.

PROTECTIVE ACTIVITY OF CALCIUM ENTRY BLOCKERS AGAINST OUABAIN INTOXICATION IN ANESTHETIZED GUINEA-PIGS

Summary: Several studies have suggested a central role for calcium in the pathogenesis of digitalis-induced arrhythmias. To test this hypothesis, the effects on ouabain-induced arrhythmia of intraarterial pretreatment with the calcium entry blockers nifedipine, flunarizine, verapamil, diltiazem, and bepridil, the calcium entry promotor Bay K 8644, and CaCl2 were compared with those of the currently applied digitalis antidotes phenytoin and lidocaine in urethane-anesthetized (1.5 g/kg i.p.) guinea pigs. Pretreatment with nifedipine (0.03 and 0.1 mg/kg), flunarizine (1 and 3 mg/kg), and phenytoin (10 mg/kg) doubled the time (from 10–20 to 20–40 min) required to provoke toxic ECG changes. Verapamil, diltiazem, and bepridil caused a slight but significant reduction of ouabain toxicity. Pretreatment with CaCl2 (10 mg/kg) enhanced all toxic effects of ouabain. None of the abovementioned pretreatments as such changed the ECG parameters. Bay k 8644 (0.03 and 0.1 mg/kg) enhanced the effects of ouabain on ventricular rhythm, but abolished the ouabain-induced impairment of AV conduction. Bay k 8644 as such increased heart rate (from 318 ± 11 to 376 ± 6 beats/min at 0.1 mg/kg) and shortened the PR interval. The negative inotropic effects of the calcium entry blockers were quantified in electrically paced (3 Hz) guinea pig isolated left atria 15 min after pretreatment with ouabain (3 x 10-7 M). The rank order of potency for the negative inotropic effect was nifedipine > verapamil > bepridil > diltiazem > flunarizine. In conclusion, nifedipine, flunarizine, and phenytoin showed obvious and equally effective protection against ouabain-induced arrhythmia. Because of the absence of severe cardiovascular side effects, flunarizine and related compounds may offer new therapeutic possibilities in the treatment of intoxication with cardiac glycosides.

Calcineurin inhibits desensitization of cloned rat 5-HT1C receptors

SummaryFunctional responses to stimulation of rat 5-HT1C receptors expressed in A9 cells were studied using whole cell voltage clamp recording technique. Stimulation of 5-HT1C receptors with serotonin (5-HT) evoked calcium-dependent outward currents of 109 pA in cells clamped at — 50 mV. Pretreatment with the protein kinase C (PKC) activator phorbol myristic acetate (PMA) reduced the 5-HT-induced current amplitude by 46% of the control value. Inclusion of inositol triphosphate (IP3) in the pipette solution induced an outward current of 84 pA. The IP3-induced response was not affected by 60 min pretreatment with PMA. In the presence of the PKC antagonist calphostin C, 60 min treatment with PMA (10−6 mol/1) reduced the 5-HT response only by 8%. In cells preincubated with PMA, injection of the calcium/calmodulin dependent serine proteinphosphatase calcineurin gradually increased the 5-HT-induced responses by 34%. In A9 cells which were incubated 24 h with the 5-HT1C receptor agonist meta chlorophenylpiperazine hydrochloride (mCPP), 5-HT-induced responses were reduced by 23% of the vehicle pretreated control value. Injection of calcineurin in mCPP treated cells enhanced the 5-HT-induced response by 24%.The results suggest that in A9 cells rat 5-HT1C receptors are desensitized after phosphorylation by PKC. This desensitization can be counteracted by calcineurin-induced: dephosphorylation.

CHARACTERIZATION OF FUNCTIONAL-RESPONSES IN A9-CELLS TRANSFECTED WITH CLONED RAT 5-HT1C RECEPTORS

SummaryFunctional responses to stimulation of rat 5-HT1C receptors expressed in A9 cells were studied using whole cell voltage clamp and calcium recording techniques. Stimulation of 5-HT1C receptors evoked outward currents clamped at −50 mV. The outward currents were reduced when GTP was excluded from the intracellular recording solution or when GDR-β-S was added.8-Bromo cyclic AMP (5 mmol/l) neither produced an effect per se nor affected the 5-HT-induced outward current in A9 cells, thus excluding CAMP as a second messenger involved in 5-HT1C receptor activation. Phorbol myristic acetate (PMA; 10 μmol/l) did not affect the electrical activity of the transfected A9 cells but reduced the 5-HT-induced current amplitude to 71±9% of the control value (n = 12). This indicates that activation of protein kinase C does not play a direct role in the 5-HT-induced response in these cells.The 5-HT induced currents mainly involved potassium ions, although a small contribution of chloride ions was also observed. The 5-HT-induced current was inhibited by the K+ channel blocking agents tetraethylammonium (1 mmol/l), apamin (0,5 μmol/l) and 4-aminopyridine (5 mmol/1). The 5-HT-induced currents recorded at −50 mV were unaffected by removal of extracellular calcium, but inclusion of the calcium chelator BAPTA (5 mmol/l) in the intracellular solutions abolished the current. Measurement with the calcium indicator Fluo-3 revealed a 5-HT-induced increase in intracellular calcium which was not affected by removal of extracellular calcium but declined after repeated stimulation.Determinated of pD2 and KB values of several 5-HT ligands using Fura-2 calcium measurements confirmed the pharmacology of the 5-HT1C receptor.The results show that cloned rat 5-HT1C C receptors expressed in A9 cells activate a calcium-dependent potassium conductance.

Cholinergic Receptors in the Extraocular Rectus Muscle of the Rabbit

The cholinergic receptors in rabbit isolated rectus muscle preparations were characterized by means of cholinergic agonists and antagonists. The concentration-dependent contraction induced by acetylcholine, carbachol or oxotremorine remained uninfluenced by atropine (10(-5) M), whereas the nicotinic receptor antagonist hexamethonium caused a parallel shift of the concentration-response curve. The agonists pilocarpine, methacholine and aceclidine, which are selective muscarinic receptor stimulants, did not cause contraction of the muscle preparation. However, the nicotinic receptor stimulant 1,1-dimethyl-4-phenylpiperazine iodide (DMPP) caused contraction similar to that induced by acetylcholine. DMPP-induced contractions were inhibited by hexamethonium, causing a parallel rightward shift of the concentration-response curve. This shift could be quantified by means of a Schild plot, indicating competitive antagonism with a PA2 value of 4.63 for hexamethonium. Atropine when applied together with hexamethonium did not cause a further shift of the concentration-response curve. The present results clearly indicate that the cholinergic receptors which mediate contraction in the rabbit isolated extraocular muscle preparation belong to the nicotinic subtype.

HEMODYNAMIC-EFFECTS OF DIFFERENT CALCIUM-ANTAGONISTS IN THE HEART-LUNG PREPARATION OF THE GUINEA-PIG

The present study was performed with the aim to demonstrate and quantify the influence of several different calcium antagonists (CA) on hemodynamic parameters in the guinea pig heart lung preparation (HLP). In paced HLP the following parameters were recorded: dp/dt max; cardiac output (CO); left ventricular pressure (LVP), and aortic pressure (AoP). In separate experiments the influence of the CA on heart rate (HR) was established in spontaneously performing HLP. All CA studied reduced or depressed dp/dt max, CO, LVP, AoP and HR. Nifedipine and verapamil showed the strongest depressant influence on dp/dt max and CO, whereas diltiazem caused a moderate reduction of these parameters. Lidoflazine, flunarizine and bepridil proved considerably less potent than nifedipine, verapamil and diltiazem. Bepridil proved least potent with respect to the influence on LVP and AoP. The strongest reduction on HR was caused by nifedipine greater than verapamil greater than diltiazem, and to a lesser degree by lidoflazine. Bepridil and flunarizine only caused a mild reduction of HR. From the calculated ratio EC20(HR)/EC20(dp/dt max) it is obvious that nifedipine, verapamil and bepridil display a much stronger influence on contractility than on HR. dp/dt max proved the most sensitive indicator for contractility in the HLP as used in our experiments.