Veronique Fardin

Characterization of a Human NK1 Tachykinin Receptor in the Astrocytoma Cell Line U 373 MG

Abstract: The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3H]substance P binding sites were present on cell homogenates, whereas [3H]neurokinin A or [3H]‐senktide binding sites were absent. The binding was saturable and reversible. The binding of [3H]substance P was inhibited by very low concentrations of [L‐Pro9]substance P and [Sar9,Met(O2)11]substance P; septide was ∼ 1,000‐fold less potent. The most potent peptide antagonist was trans‐4‐hydroxy‐1‐(1 H‐indol‐3‐ylcarbonyl)‐L‐prolyl‐N‐methyl‐N‐(phenylmethyl)‐L‐tyrosineamide. The rank order of potency for the nonpeptide antagonists was (S,S)‐CP 96,345 > (±)‐CP 96,345 > (±)‐2‐chlorobenzylquinuclidinone > (R,R)‐CP 96,345 > RP 67580 > RP 68651. In [3H]‐inositol‐labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor.

Analgesia Produced by Stimulation of the Periaqueductal Gray Matter: True Antinoceptive Effects Versus Stress Effects

Since Reynold’s initial report years ago (1969) revealed that during and after periaqueductal gray matter (PAG) stimulation rats could support severe, painful interventions such as laparotomies, the “stimulation produced analgesia phenomenon” (SPA) has been extensively studied in the rat, cat and monkey (see reviews in Liebeskind et al., 1976; Mayer and Price, 1976; Basbaum and Fields, 1978; Mayer, 1979; Besson et al., 1981; Basbaum and Fields, 1984; Oliveras and Besson, 1988). Furthermore, SPA from central gray (anatomically including the caudal periventricular hypothalamic region and the PAG) has been used to relieve severe intractable pain in human patients (Adams, 1976; Gybels et al., 1976; Hosobuchi et al., 1977; 1979; Richardson and Akil, 1977a,b). However, after an initial rush of enthusiasm by neurosurgical teams involved in this pain treatment, the use of central gray deep brain stimulation became controversial (Gybels et al., 1976; Meyerson, 1988). These controversies are justifiable in light of certain behavioral observations: the PAG and the central gray are complex regions involved in diverse functions and states including not only analgesia but also “intense emotion” (very unpleasant to intolerable sensations) when electrically stimulated in both human beings and animals.

Characterization of NK1 and NK2 tachykinin receptors in guinea-pig and rat bronchopulmonary and vascular systems.

1 NK1 and NK2 tachykinin receptors were characterized in guinea‐pig and rat bronchopulmonary systems and in the vasculature of the rat by use of radioligand binding and/or functional studies. 2 The radioligands for NK1 and NK2 receptors ([3H]‐SP and [3H]‐pNKA, respectively) did not label tachykinin receptors in homogenates of rat lungs or bronchi. In contrast, in the guinea‐pig, [3H]‐SP bound with high affinity to these tissues (KD = 0.23 ± 0.08 nm and 0.34 ± 0.05 nm, for lungs and bronchi, respectively). The total number of binding sites was 4.6 fold greater in bronchus (Bmax = 135 ± 27 fmol mg−1 protein) than in lung homogenates (Bmax = 29.3 ± 0.1 fmol mg−1 protein). Furthermore, this binding was markedly displaced by CP‐96,345 (pKi = 9.5 ± 0.1) and RP 67580 (pKi = 7.6 ± 0.1), antagonists of NK1 receptors, slightly displaced by SR 48968 (pKi = 6.6 ± 0.1), but not affected by actinomycin D or L‐659,877, antagonists of NK2 receptors. Specific binding of [3H]‐pNKA, detected in guinea‐pig bronchi (KD = 5.2 ± 0.1 nm, and Bmax = 203 ± 19 fmol mg−1 protein) but not in lungs, was similarly (40 to 53%) displaced by RP 67580 (1 μm), CP‐96,345 (10 and 100 nm) or SR 48968 (10 and 100 nm). The displacement approximately doubled (87 to 91%) when SR 48968 (10 nm) was combined with either RP 67580 (1 μm) or CP‐96,345 (10 nm), but not when RP 67580 was combined with CP‐96,345. 3 In urethane‐anaesthetized guinea‐pigs, i.v. injections of the NK1 receptor agonists SP, [Pro9]‐SP, [Sar9, Met(O2)11]‐SP and septide, as well as the NK2 receptor agonists NKA and [Lys5, MeLeu9, NLeu10]‐NKA(4–10) (0.1–10 μg kg−1, i.v.), dose‐dependently increased lung inflation pressure. The most potent of these peptides were septide and [Lys5, MeLeu9, NLeu10]‐NKA(4–10) (EC50 = 0.38 ± 0.07 and 0.07 ± 0.02 μg kg−1, respectively). Interestingly, septide was 130 fold less potent than SP in displacing [3H]‐SP from its binding sites in the guinea‐pig lung, whereas it was 14 fold more potent than SP as a bronchoconstrictor. RP 67580 (0.3–5 mg kg−1, i.v.) and CP‐96,345 (0.01–3 mg kg−1, i.v.) dose‐dependently reduced the bronchoconstriction produced by the NK1 receptor agonists. Conversely, the NK2 receptor antagonists actinomycin D (1–10 mg kg−1, i.v.) and SR 48968 (0.03–0.3 mg kg−1, i.v.) inhibited specifically the responses induced by NK2 receptor agonists. 4 In pentobarbitone‐anaesthetized rats, the NK1 and NK2 receptor agonists (0.01–4 μg kg−1, i.v.) produced dose‐dependent hypotensive responses. The order of potency was SP = [Sar9, Met(O2)11]‐SP = [Pro9]‐SP > septide = NKA >[Lys5, MeLeu9, NLeu10]‐NKA(4–10). RP 67580 (0.13–0.5 mg kg−1, i.v.) and CP‐96,345 (0.5–2 mg kg−1, i.v.) antagonized in a dose‐related manner (20 to 64%) the vascular effects of both NK1 and NK2 receptor agonists, whereas actinomycin D (3 mg kg−1, i.v.) and SR 48968 (2 mg kg−1, i.v.) did not. RP 67580 was approximately 4 times more potent than CP‐96,345. 5 These studies indicate that NK1 and NK2 receptors are both present in the guinea‐pig bronchopulmonary system whereas only NK1 receptors are detectable in the rat vasculature under our experimental conditions. Furthermore, NK1 receptors in the guinea‐pig bronchopulmonary system are pharmacologically distinct from those present in the rat vascular system, since both agonist potencies and antagonist affinities differ between the two species.

Species differences between [3h] substance P binding in rat and guinea-pig shown by the use of peptide agonists and antagonists

The affinities of various substance P agonists and antagonists for NK1 receptors in rat and guinea-pig tissues were compared. Striking species differences were observed. Both septide and [D-Pro4,D-Trp7,9]SP-(4-11) possessed much higher affinity for sites in the guinea-pig (brain and ileum) than for sites in the rat brain. These results could be explained by differences in the structure of the NK1 receptor according to the species, although the existence of various subtypes of NK1 binding sites in the two species cannot be excluded.

Antinociceptive action following microinjection of methionine-enkephalin in the nucleus raphe magnus of the rat

The analgesic effect of the microinjection of low doses of methione-enkephalin (20 micrograms in 0.5 microliter) into the caudal brain stem of the unrestrained rat was investigated by means of one vocalisation test. Immediate short duration significant increases in threshold (21%) were seen from sites in the nucleus raphé magnus (NRM). Delayed effects were seen from sites immediately adjacent to this nucleus; sites more lateral produced no significant effect. These results lend further support to the postulated role of NRM in antinociception.

NK-1 tachykinin receptor in rat and guinea pig brains: Pharmacological and autoradiographical evidence for a species difference

The affinities of a large variety of peptide or nonpeptide tachykinin analogues were determined on membranes from rat and guinea pig brains using the selective NK-1 radioligand 3H-[Pro9]SP. Nonpeptide antagonists clearly revealed a species difference; (+/-)CP-96,345 was more potent in the guinea pig, while RP 67580 was found to be a better competitor of 3H-[Pro9]SP binding to rat brain membranes. This was confirmed on brain slices by autoradiography. Numerous brain structures were analyzed by optical densitometry. From these data, a heterogeneity of NK-1 binding sites among different structures can be excluded in both rat and guinea pig brains.

Variations in Plasma Levels of Substance P and Effects of a Specific Substance P Antagonist of the NK1 Receptor on Preovulatory LH and FSH Surges and Progesterone Secretion in the Cycling Cynomolgus Monkey

These studies investigated the role of substance P (SP) in the regulation of the hypothalamic-pituitary-ovarian axis in cynomolgus monkeys with normal menstrual cycles. Plasma concentrations of SP were determined in blood samples taken every morning in normally menstruating cynomolgus monkeys throughout the menstrual cycle. There was a significant decreasing linear trend of SP during the follicular phase (cycle day –13 to day 0) and a significant inverse relationship between SP plasma values and plasma 17β-estradiol (E2) values from day –13 to day 0 of the adjusted cycle. Correspondingly, SP area under the curve was significantly greater during the follicular phase than the luteal phase. In a second experiment, plasma concentrations of E2, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone and length of cycles were measured after five daily intragastric administrations (10 mg/kg) of an NK1 receptor (SP receptor) antagonist (RPR 100893; 10 mg/kg) initiated after serum E2 concentrations had exceeded 125 pg/ml. There was a statistically significant reduction in the amplitude (41% of control) and the area under the curve (37% of control) of the preovulatory LH surge. In addition, there was a reduction of the duration of the LH surge (3 ± 0.1 days in controls vs. 2.1 ± 0.2 days in treated animals). The present results show for the first time that there are significant variations in plasma levels of SP, with a strong negative correlation with serum levels of E2 during the follicular phase of the cynomolgus monkey, and that endogenous SP has a potentiating role in the interactive hypothalamo-anterior-pituitary mechanisms which lead to the preovulatory LH and FSH surges during the menstrual cycle in the monkey.

In vitro and in vivo evidence for a tachykinin NK1 receptor antagonist effect of vapreotide, an analgesic cyclic analog of somatostatin

Vapreotide, a long-acting somatostatin analog, possesses an analgesic effect. The purpose of this work was to determine a tachykinergic involvement. Vapreotide reduced substance P-induced biting and scratching in mice. This inhibitory effect of substance P action was confirmed by experiments performed on the bronchial apparatus of guinea-pigs known to possess tachykinin NK1 and NK2 receptors. (i) Vapreotide reduced the substance P-induced plasmatic exudation. (ii) It inhibited selectively the tachykinin-dependent second contractile phase induced by electrical field stimulation of isolated bronchi. (iii) It shifted to the right the concentration-effect curve of substance P-induced contraction of isolated main bronchi. The peptide displaced [3H]substance P (IC50 = 3.3 +/- 1.8 x 10(-7) M) from guinea-pig bronchial tachykinin NK1 sites. The displacement of [125I]neurokinin A, a specific tachykinin NK2 receptor ligand, needed higher concentrations (IC50 = 4.5 +/- 0.6 x 10(-6) M). It is concluded that vapreotide possesses an antagonist activity on guinea-pig tachykinin NK1 receptors; the involvement in its analgesic action is discussed.

Electrographic studies of the effects of RP 60180, a novel kappa agonist, on the photosensitive baboon Papio papio

1. The authors describe here the effects of intravenous administration of RP 60180, a novel kappa agonist, on conscious baboons of the species Papio papio, which spontaneously present photically induced epileptic responses. 2. Animals (n = 2) were chronically implanted with epidural recording electrodes and tested whilst seated in a primate chair. The electrocorticogram (ECoG) and electrocardiogram (ECG) were recorded during a control period of at least 30 minutes before the injection of RP 60180 (1 to 4.5 mg/kg i.v.) and immediately afterwards. 3. Qualitatively, up to the dose of 4.5 mg/kg i.v., RP 60180 did not modify ECoG background in term of paroxysmal activity in comparison with that observed during the control period. It did not cause any manifest focal or generalized seizure discharges, nor did it consistently enhance or reduce photically induced myoclonic responses. 4. From the dose of 1 mg/kg i.v., RP 60180 slowed ECG frequency. This effect, which lasted for about 30 minutes post-injection, was most often seen at the higher doses. 5. In another set of experiments, one baboon received the kappa agonist U-50488 (a benzacetamide derivative of spiradoline) at 1 and 3 mg/kg i.v. U-50488, at 3 and to a lesser degree at 1 mg/kg i.v., induced paroxysmal bursts of slow wave ECoG activity and a slowing of the ECG. These effects lasted about 1 hour post-drug administration. During this period, we observed spontaneous vocalization, as if the animal were complaining, as well as shaking.