Francis Rioux

Autoradiographic distribution of [3H]neurotensin receptors in rat brain: Visualization by tritium-sensitive film

[3H]Neurotensin ([3H]NT) binds specifically to a single class of binding sites on slides-mounted sections of rat brain 1Kp = 5.1 nM; Bmax = 16.2 fmol/mg tissue). Bound [3H]NT can be displaced by nonradioactive NT and a series of its fragments and analogues with relative potencies that correlate closely (r = 0.89; p less than 0.01) to their potencies in the rat stomach strip bioassay. These results suggest that NT receptors are similar in both systems. [3H]NT binding sites were visualized by using tritium-sensitive LKB film analysed by computerized densitometry. [3H]NT receptors are highly concentrated in the external layer of the olfactory bulb, in the rhinal sulcus, in certain nuclei of the amygdala, in the substantia nigra, zona compacta and in the ventral tegmental area. The high density of [3H]NT receptors in the last two areas suggest an interaction between NT and brain dopaminergic systems such as the nigrostriatal and the mesolimbic pathways.

The vasoconstrictor effect of neuropeptide Y and related peptides in the guinea pig isolated heart

Neuropeptide Y (NPY) infusions into isolated, perfused, spontaneously beating hearts of guinea pigs elicited concentration-dependent increases of myocardial perfusion pressure and decreases of myocardial tension, but no consistent changes of heart rate. The increase of perfusion pressure caused by NPY (attributed to a constrictor effect on coronary vessels) was not affected by atropine, prazosin, yohimbine, propranolol, cimetidine, diphenhydramine, indomethacin or a mixture of methysergide and morphine. However, it was reduced by verapamil, a Ca2+ antagonist. Deletion of the N-terminal amino acid Tyr1 from the NPY molecule caused a 12-fold reduction of NPY potency as a coronary constrictor. Further shortening of the NPY molecule by removal of sequence Tyr1 through Glu15 or Tyr1 through Ala18 caused major losses of potency without detectable reduction of intrinsic activity. The results suggest that the constrictor effect of NPY on guinea pig coronary vessels results from a direct effect on vascular smooth muscle cells, is mediated by specific receptors and is likely to involve the participation of extracellular calcium ions. The results also suggest that the chemical groups responsible for the vasoconstrictor effect of NPY in guinea pig hearts might be scattered in the C-terminal end of the peptide.

The stimulatory effects of neurotensin and related peptides in rat stomach strips and guinea-pig atria

1 The stimulatory effects of neurotensin (NT) and several NT fragments were evaluated in two pharmacological preparations: rat stomach strips and isolated spontaneously beating atria of guinea‐pigs. 2 In rat stomach strips, NT elicited a dose‐dependent contractile effect in concentrations varying between 1.3 × 10−9 and 5.4 × 10−7 M. 3 The contractile effect of NT (1.3 and 5.4 × 10−8 m) in this tissue was not modified by atropine (3.4 × 10−7 m), methysergide (2.0 × 10−6 m), a mixture of cimetidine (8.0 × 10−6 m) and diphenhydramine (7.8 × 10−6 m), indomethacin (1.4 × 10−5 m), 8‐Leu‐angiotensin II (1.0 × 10−6 m), glucagon (2.0 × 10−6 m) or somatostatin (3.0 × 10−7 m). 4 Rat stomach strips desensitized by bradykinin (6.1 × 10−6 m) or substance P (7.4 × 10−6 m) maintained their sensitivities to NT (1.3 and 5.4 × 10−8 m). 5 In guinea‐pig atria, NT produced a dose‐dependent positive inotropic action in concentrations varying between 5.4 × 10−10 and 2.7 × 10−7 m. 6 The inotropic effect of NT (2.7 × 10−9 m) was not influenced by methysergide (2.8 × 10−6 m), atropine (3.4 × 10−7 m), practolol (1.5 × 10−5 m), 8‐Leu‐angiotensin II (1.0 × 10−6 m), or indomethacin (1.4 × 10−5 m), but it was reduced by 37% by cimetidine (4.0 × 10−5 and 2.0 × 10−4 m). A combination of cimetidine (4.0 × 10−5 m) and diphenhydramine (3.9 × 10−6 m) did not produce a greater inhibition of NT than cimetidine alone. 7 Atria desensitized by bradykinin (6.1 × 10−6 m) or glucagon (2.0 × 10−6 m) maintained their sensitivities to NT (2.7 × 10−9 m). Substance P was inactive both as an agonist or antagonist of NT. 8 These results suggest the existence of specific NT receptors in rat stomach strips and guinea‐pig atria. 9 The data derived from our structure‐activity study suggest that the minimum structure required for the full stimulation of NT receptors in these two preparations is H‐Arg9‐Pro10‐Tyr11‐Ile12‐Leu13‐OH. The sequence PyroGlu1‐Leu2‐Tyr3‐Glu4‐Asn5‐Lys6‐Pro7‐Arg8‐ and the amino acids Ile12 and Leu13 appear to contribute mainly to the affinity or binding of NT to its receptor. The chemical groups responsible for the full activation (intrinsic activity) of NT receptors seem to be located in the sequence ‐Arg9‐Pro10‐Tyr11.

The hypotensive effect of centrally administered neutrotensin in rats

We have evaluated the cardiovascular effects of intracerebroventricular (i.c.v.) injections of neurotensin (NT) in pentobarbital-anesthetized rats. In most animals, the i.c.v. injection of NT (5.4, 10.8 and 16.2 nmol/rat) induced a dose-dependent fall of the arterial blood pressure. This effect was usually rapid in onset (30-60 sec) and of short duration (approximately 1-4 min). It was not preceded nor accompanied by any significant alteration of the heart rate. In about 25% of the animals, the vasodepressor effect of i.c.v. injections of NT was long lasting (30-45 min). Conscious rats were much less sensitive than anesthetized animals. The hypotensive effects of intravenously (i.v.) administered NT was fully maintained in animals made tolerant to the hypotensive effect of centrally administered NT. Similarly, the animals made unresponsive to i.v. injections of NT either by repeated i.v. injections of NT (e.g. tachyphylaxis) or by a chronic treatment with compound 48/80, still responded normally to centrally administered NT. The results suggest the existence of at least two anatomically distinct sites of action through which NT can induce hypotension in rats. One appears to be located in the periphery and the other, in the central nervous system.

Pharmacological characterization of neurotensin receptors in the rat isolated portal vein using analogues and fragments of neurotensin

The contractile effects of the tridecapeptide neurotensin (NT) and several NT fragments and analogues were evaluated and compared in the rat isolated portal vein. The removal of the sequence pGlu1-Leu2-Tyr3-Glu4-Asn5-Lys6-Pro7 produced practically no change in the myotropic activity of NT while the deletion of Leu13 or the last 3 C-terminal amino acids (e.g. Tyr11, Ile12 and Leu13) gave compounds with very low agonist activity (NT(1-12)) or devoid completely of affinity and intrinsic activity (NT(1-10)). Replacing Tyr11 with Ala, Leu, D-Tyr or D-Phe markedly decreased the stimulant effect of NT but did not confer to the molecule antagonistic properties. On the other hand, the substitution of Try11 with D-Trp or Tyr(Me) gave NT analogues which behave as specific and competitive antagonist of the contractile effect of NT in the portal vein. pA2 values of [D-Trp11]-NT and [Tyr(Me)11]-NT measured in the venous preparation were similar to those found in the coronary vasculature of the rat. Taken all together, these results suggest that: (1) the minimum structure required for the full expression of the myotropic activity of NT in the rat portal vein is -Arg9-Pro10-Tyr11-Ile12-Leu13-OH; (2) Tyr11 appears to be closely involved in the process of NT receptor activation since its replacement with D-Trp or Tyr(Me) produced specific and competitive antagonist of NT; (3) the receptors mediating the contractile effect of NT in the rat portal vein appear to be pharmacologically similar to those found in the coronary vessels of the rat. The possibility for the existence of different types of NT receptor in other tissues is discussed.

Compound 48/80 inhibits neurotensin-induced hypotension in rats

Abstract The intravenous injection of neurotensin (NT) (0.4 and 1.1 nmoles/kg) produced dose-dependent hypotensive effects in pentobarbital anesthetized rats. The acute or chronic administration of compound 48/80, a well known mast cell depletor, completely abolished the hypotensive effect of low to medium doses of NT and unmasked the previously unknown hypertensive effect of high doses (4.0 nmoles/kg) of NT. This hypertensive effect was significantly reduced by infusing the animals with [D-Trp 11 ]-NT a selective antagonist of NT. The hypotensive action of NT in control rats was also significantly reduced by pretreating the animals with disodium cromoglycate, an antiallergic drug which is believed to stabilize mast cells membranes, or with a mixture of azatadine and methysergide. The results suggest the participation of histamine, serotonin and possibly other endogenous vasoactive substances, to the hypotensive action of NT in rats. The possible origin of these mediators is discussed.

Recombinant human hemoglobin (rHb1.1) selectively inhibits vasorelaxation elicited by nitric oxide donors in rabbit isolated aortic rings

We investigated the effect of a genetically engineered recombinant human hemoglobin (rHb1.1), specially designed to be used as a blood substitute, on the ability of various well-known vasodilators to relax the rabbit isolated aortic rings precontracted with the alpha-adrenoceptor agonist phenylephrine (PE) or with KCl (for nifedipine only). The vasorelaxant effects of nitroglycerin (NTG) and of sodium nitroprusside (SNP), two nitrovasodilators whose effects are mediated by nitric oxide (NO), were inhibited in a concentration-dependent manner by rHb1.1 (1.5 and 15 microM). Those elicited by isoproterenol, papaverine, histamine, adenosine, atriopeptin II, hydralazine, nifedipine, and cromakalim were comparatively little affected or not affected by rHb1.1 (15 microM). The ability of captopril to inhibit the vasoconstrictor action of angiotensin I (AT1) in the rabbit aortic rings was not reduced by rHb1.1 (15 microM). Our results suggest that rHb1.1 shares with purified human Hb the ability to inhibit selectively the vasorelaxant effect of NO-releasing substances such as NTG and SNP. Because the targeted plasma concentration of rHb1.1, when used as a blood substitute, is greater (approximately 50 times) than the highest concentration of rHb1.1 used in this study, significant drug interactions can be predicted between NO donors and rHb1.1.

Pharmacological studies of neurotensin, several fragments and analogues in the isolated perfused rat heart

Neurotensin (NT) induced a dose-dependent increase of the coronary perfusion pressure (CPP) in the isolated perfused rat heart. This effect was not modified by pretreating the organ with methysergide (8.5 x 10(-6) M), atropine (3.4 x 10(-6) M), a mixture of phentolamine (3.1 x 10(-6) M) and practolol (1.5 x 10(-5) M), 8-leucine-angiomine (2.9 x 10(-5) M) thus suggesting the existence of specific NT receptors in the coronary vessels of rat. The structure-activity studies performed using several NT fragments and analogues in the isolated perfused rat heart led us to the following conclusions: (1) the minimum structure required for the full expression of the biological activity of NT is H-Arg9-Pro10-Tyr11-Ile12-Leu13-OH; (2) the amino acid Tyr in position 11 appears to play a key role in the process of NT receptor activation. The replacement of Tyr11 with Tyr(Me) gave a compound which inhibits selectively the increase in coronary perfusion pressure induced by NT, but still exhibits some NT-like activity, specially when used in concentrations higher than 10(-6) M. [Tyr(Me)11]NT did not antagonize the stimulant effects of NT in rat stomach strips and guinea pig atria, thus suggesting that the receptors mediating the constrictor effect of NT in coronary vessels of the rat are pharmacologically different from those subserving the stimulant effect of NT in rat stomach strip and guinea pig atria.

Selective blockade of neurotensin-induced coronary vessel constriction in perfused rat hearts by a neurotensin analogue

[D-Trp11]-NT, an analogue of neurotensin (NT) in which Tyr11 was replaced with D-Trp, was found to antagonize selectively NT-induced coronary vessel constriction in perfused rat hearts, in concentrations varying between 1.3 x 10(-7) and 1.1 x 10(-6) M. Higher concentrations of [D-Trp11]-NT displayed NT-like activity. In rat stomach strips and guinea pig atria, [D-Trp11]-NT exhibits full intrinsic activity, markedly reduced affinity for NT receptors, but no inhibitory effect against NT. These results suggest that the receptors mediating the constrictor action of NT in the coronary vessels of rat hearts are pharmacologically distinct from those subserving the stimulant effects of NT in rat stomach strips and guinea pig atria.

The contractile effect of bombesin, gastrin releasing peptide and various fragments in the rat stomach strip

The contractile activity of bombesin (BB) and related peptides including the gastrin releasing peptide (GRP) was evaluated in the rat stomach strip. BB and GRP were found to elicit concentration-dependent contractile effects in concentrations varying between 5.0 X 10(-10) and 5.0 X 10(-7) M. EC50 values of BB and GRP were 6.5 X 10(-9) and 10(-8) M, respectively. The contractile effect of BB (1.5 X 10(-8) M) was not modified by a mixture of antagonists containing atropine (1.7 X 10(-6) M), mepyramine (1.2 X 10(-6) M), methysergide (1.4 X 10(-6) M), phentolamine (1.8 X 10(-6) M), propranolol (1.9 X 10(-6) M) and indomethacin (7.0 X 10(-6) M), nor by the nerve paralyzing drug tetrodotoxin (1.6 X 10(-6) M). These results suggest the existence of BB receptors in the rat stomach strip. The results also suggest that the contractile effects of BB in this tissue result from a direct effect on the smooth muscle cell. The data derived from our structure-activity study indicate that deletion of the N-terminal sequence pGlu1-Gln2-Arg3-Leu4-Gly5- from the BB molecule causes practically no loss of affinity and intrinsic activity. Further shortening of BB gives rise to a gradual reduction of both parameters. Residual contractile activity could still be observed with the tetra- and pentapeptide BB-(11-14) and BB-(10-14) at 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)