Domenico Regoli

Characterization of the peripheral action of neurokinins and neurokinin receptor selective agonists on the rat cardiovascular system

SummaryThe effects on mean arterial pressure (MAP) and heart rate (HR) of increasing doses (0.65–65 nmol/kg) of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were measured after intravenous (i. v.) injection in urethane anaesthetized rats. Neurokinins (NKs) elicited a vasodepressor effect with the following rank order of potency: SP (100%) > NKB (17.5%) > NKA (10%). The two undecapeptide NK-1 selective agonists, [Pro9, Met(O2)11]SP (787%) and [Sar9, Met(O2)11]SP (697%), evoked a significantly (P < 0.05) greater vasodepressor response than SP, while the potency of the octapeptide NK-1 selective agonist [β-Ala4, Sar9, Met(O2)11]SP (4–11) (316%) was not significantly different from SP. Conversely, the NK-2 selective agonist NKA (4–10) (<2%) caused only a small effect. The vasodepressor effect elicited by [MePhe7]NKB (112%) and [β-Asp4, MePhe7]NKB (4–10) (92%), two NK-3 selective agonists, were not significantly different from that of SP. Senktide (1,095%) is the most potent NK-3 agonist, and is significantly (P < 0.01) more potent than SP. No cross-desensitization, of the vasodepressor response, was observed between NK-1 and NK-3 selective agonists. I. V. injection of 32.5 nmol/kg of NKA, NKA (4–10) and [β-Ala4, Sar9, Met(O2)11]SP (4–11) raised HR, while NKB and the NK-3 selective agonists produced a rapid and marked bradycardia. SP and the two undecapeptide, NK-1 selective agonists, produced an initial increase in HR and a latent long-lasting bradycardia. The bradycardia elicited by [Sar9, Met(O2)11]SP (32.5 nmol/kg) was blocked by methylatropine, hexamethonium, indomethacin and by treatment with capsaicin or compound 48/80. Although the bradycardia elicited by [β-Asp4, MePhe7]NKB (4–10) (32.5 nmol/kg) was also blocked by hexamethonium, methylatropine, and by bilateral vagotomy, it remained unaffected after indomethacin, or in rats pretreated with either capsaicin or compound 48/80. The drop in MAP produced by the NK-1 and NK-3 agonists were reduced by hexamethonium, methylatropine and bilateral vagotomy (NK-3 agonist), but remained unaffected by indomethacin, capsaicin, and compound 48/80. The tachycardia to NKA (4–10) (65 nmol/kg) was blocked entirely by sotalol or metoprolol and potentiated by hexamethonium. Guanethidine and bilateral adrenalectomy (48 h) failed to affect the tachycardia induced by the agonist, whereas the combination of both treatments abolished the response. Rats sympathectomized with 6-hydroxydopamine (48 h) reduced the increase in HR to NKA (4–10) only at 1 min post-administration. These results support the hypothesis that the vasodepressor action of SP is mediated by the direct activation of NK-1 receptors on vascular endothelium. NKs can accelerate HR by releasing catecholamines through activation of NK-1 and/or NK-2 receptors, on noradrenergic fibers and adrenal chromaffn cells. Furthermore NKs can exert a depressor effect on the cardiovascular system by producing a vagal reflex (Bezold-Jarisch reflex), following either the activation of NK-3 receptors located on capsaicin-insensitive sensory fibers, or by promoting the release of prostaglandins from mast cells (via a non-receptor mechanism) which in turn will stimulate capsaicin-sensitive sensory fibers.

Capillary permeability induced by intravenous neurokinins

SummaryThe effects on plasma extravasation of three increasing doses from 6.5 pmol to 650 nmol/kg of substance P (SP), SP fragments, neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were assessed in three cutaneous tissues (skin of hind paws, dorsal skin and ears) by intravenous (i.v.) administration in the pentobarbitone anaesthetized rat. Dose-dependent increases in plasma extravasation were observed with the following rank orders of potency (SP > NKA > NKB) for neurokinins and (SP > [p-Glu6]SP(6–11) > SP(4–11) > [p-Glu5]SP(5–11) > SP(7–11)) for C-terminal SP fragments. The metabolically stable SP analogue [p-Glu5, McPhe8, Sar9]SP(5–11) was slightly more potent than [p-Glu5]SP(5–11). The N-terminal fragments SP(1–4), SP(1–7) and SP(1–9) were inactive up to 650 nmol/kg. The NK-1 receptor selective agonists [Sar9, Met(O2)11]SP and [β-Ala4, Sar9, Met (O2)11]SP(4–11) were more potent than the NK-2 ([Nle10]NKA(4–10)) and NK-3 ([MePhe7]NKB and β-Asp4, McPhe7NKB(4–10)) receptor selective agonists.Plasma extravasation induced by SP (6.5 nmol/kg) was unchanged in the presence of atropine, methysergide, diphenhydramine or during the i.v. and intra-arterial (i.a.) infusion of d-Arg0[Hyp3. d-Phe7]BK, an antagonist of bradykinin. Plasma extravasation induced by SP and [Sar9, Met(O2)11]SP was significantly reduced by indomethacin while that induced by NKA, NKB, [β-Ala4, Sar9, Met(O2)11]SP(4–11), SP(4–11) and [p-Glu6]SP(6–11) was unaffected by the cyclooxygenase inhibitor. Compound 48/80 (0.75 mg/kg), histamine (10 mg/kg) and 5-HT (10 mg/kg) caused an incrase in plasma extravasation, only the effect of compound 48/80 was abolished by indomethacin. Pretreatment with compound 48/80 prevented its own action on plasma extravasation and significantly reduced that induced by 6.5 nmol/kg of SP.These results rule out the involvement of acetylcholine (muscarinic receptors), 5-HT (5-HT1 and 5-HT2 receptors), histamine (H1 receptors) and kinins (B2 receptors) in the response to SP and indicate that the two positively charged amino acids (Arg, Lys) at the N-terminal end of the SP molecule are essential to trigger the release of prostaglandins from mast cells. This mechanism is responsible for the indirect effect of SP and related peptides on capillary permeability and does not appear to be mediated by a selective SP receptor. In addition, neurokinins may increase capillary permeability by direct activation of a NK-1 receptor type on the vascular endothelium.

Involvement of bradykinin B1 and B2 receptors in pulmonary leukocyte accumulation induced by Sephadex beads in guinea pigs.

The effects of selected bradykinin receptor antagonists on leukocyte infiltration into the lungs were studied in a model of guinea pig lung inflammation induced by the intravenous injection of Sephadex beads. The bradykinin B1 receptor antagonist, [Leu8]desArg9-BK (40 mg kg(-1) 24 h(-1)) and the bradykinin B2 receptor antagonist, DArg[Hyp3,Thi5,DTic7,Oic8]BK (code name HOE 140; 4 mg kg(-1) 24 h(-1)), administered intravenously by osmotic pumps, significantly reduced eosinophil counts by 33% and 42% in bronchoalveolar fluid, respectively. HOE 140 decreased neutrophil counts by 35%. LysLys[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK+ ++ (code name B 9858), a newly described bradykinin B1 receptor antagonist, administered intraperitoneally (1 mg kg(-1)), decreased eosinophil and neutrophil counts by 45% in bronchoalveolar fluid. D-Arg[Hyp3,Igl5,D-Igl7,Oic8]BK (code name B 9430), a non-selective bradykinin B1/B2 receptor antagonist, also administered intraperitoneally (1 mg kg(-1)), decreased eosinophil and macrophage counts by 62% and 80% in bronchoalveolar fluid. These results suggest that bradykinin B1 and B2 receptors are involved in leukocyte recruitment in our model of lung inflammation.

Characterization of spinal actions of four substance P analogues

Four substance P (SP) analogues were tested on reaction time (RT) in the tail flick test and on the decrease in RT produced by the SP homologue physalaemin. The four analogues were [D-Trp7,9,10]SP, denoted A, [D-Pro4,D-Trp7,9,Nle11]SP-(4-11), denoted B, [D-Pro2,D-Trp7,9,10]SP, denoted C and [D-Pro4,D-Trp7,9,10,Phe11]SP-(4-11), denoted D. Physalaemin alone (1.89 nmol) reduced RT. The analogue A, at 3.25 nmol, blocked the effects of physalaemin without altering basal RT. The analogues B and D, which block the action of physalaemin in peripheral tissues, had neither agonistic nor antagonistic effects in doses up to 6.5 nmol. The replacement of L-Pro2 by D-Pro2 in the analogue A yielded the analogue C, which had no antagonistic activity. All analogues produced a flaccid paralysis of the hindlimbs and the tail; this effect was inconsistent, though, occurring only in some rats, and appearing in some cases after the first administration of the analogue yet in other cases only after a subsequent administration. Because B and D are inactive in the spinal cord, our results suggest that physalaemin activates receptors in the spinal cord different from those it activates in peripheral tissues. Furthermore, because all four analogues produced a flaccid paralysis none is suitable for use as an SP antagonist in vivo in the CNS.

In vitro and in vivo pharmacology of S 16474, a novel dual tachykinin NK1 and NK2 receptor antagonist

Since tachykinins released from lung sensory nerve endings are thought to play a role in inflammatory diseases of airways via NK1 and NK2 receptors, dual tachykinin NK1 and NK2 receptor antagonists may have a great therapeutic potential. In vitro, the cyclopeptide S 16474 (cyclo-[Abo-Asp(D-Trp(Suc0Na)-Phe-N-(Me)Bzl)]) bound to both human tachykinin NK1 and NK2 receptors expressed in two lines of transfected Chinese hamster ovary cells (IC50 values 85 nM and 129 nM, respectively), while showing a poor affinity for the rat tachykinin NK1 receptor. S 16474 inhibited the contractions induced by substance P in isolated rabbit vena cava (pA2 7.0) and by neurokinin A in rabbit pulmonary artery (pA2 5.6). In vivo in anaesthetized guinea-pigs, S 16474 was found to dose dependently inhibit the bronchoconstrictions induced by intravenously administered substance P, neurokinin A and capsaicin. Plasma extravasation evoked in bronchi by endogenously released tachykinins under vagus nerve stimulation was abolished by S 16474 (10 mu mol/kg i.v.). These results demonstrate clearly that S 16474 is a tachykinin receptor antagonist exhibiting, in vitro and in vivo, a dual inhibitory effect on NK1 and NK2 receptors.

Bradykinin B2 type receptor activation regulates fluid and electrolyte transport in the rabbit kidney.

Bradykinin is an important autacoid produced in the kidney, regulating both renal function and blood pressure. In vivo studies in anesthetized rabbits, revealed that BK induced diuresis (UV), natriuresis (U(Na)V) and was not associated with renal hemodynamic changes. These diuretic and natriuretic effects were blocked by the BK-B2 antagonist HOE-140. BK also inhibits vasopressin (AVP)-stimulated water flow (L(p)) in microperfused rabbit cortical collecting ducts (rCCD), in a concentration-dependent fashion, consistent with its in vivo diuretic effects. BK-B1 antagonist Leu8-des-Arg9-BK did not alter the effect of BK on Lp, but HOE-140 completely blocked the inhibitory effects of BK on Lp. While BK did not increase [Ca2+]i in fura-2 loaded freshly microdissected rCCD, BK increased [Ca2+]i in immortalized cultured rCCD cells demonstrating different signaling mechanisms are activated by BK in microdissected versus cultured rCCD. In microperfused rCCD, neither the protein kinase C inhibitor staurosporine nor the phospholipase C (PLC) inhibitor U-73,122 attenuated the BK response arguing against activation of PLC/PKC by BK in rCCD. We conclude: (1) BK induces UV and U(Na)V by a BK-B(2) receptor; (2) BK inhibits AVP-stimulated Lp by a BK-B2 receptor suggesting that its effects on Lp are not via a PLC/PKC; (3) finally, BK raises [Ca2+]i in rCCD cells by a BK-B2 receptor mechanism.

Involvement of NK1 receptors and importance of the N-terminal sequence of substance P in the stimulation of protein secretion in rat parotid glands

Recent in vitro studies have shown that the dose-response curve of substance P on [3H]protein secretion from rat parotid glands is biphasic. Such a response could result either from the activation of tachykinin receptors or from the amphiphilic character of substance P, since it has previously been shown that the N-terminal part of substance P may play an important role in the activation of phosphoinositides in rat parotid glands. To investigate these possibilities, we studied the effects of selective NK1, NK2, NK3 receptor agonists and C-terminal fragments of substance P and neurokinin A on protein secretion from rat parotid lobules. The poor activity of NK2 (neurokinin A-(4-10) and [beta-Ala8]neurokinin A-(4-10)) as well as of NK3 ([MePhe7]neurokinin B) selective agonists allowed us to rule out a possible involvement of NK2 and NK3 receptors in the parotid gland secretory process. Conversely, the selective NK1 receptor agonist, [Sar9,Met(O2)11]substance P, reproduced the biphasic dose-response curve for [3H]protein secretion typical of native substance P. However, a biphasic response was not observed with peptides deprived of the N-terminal moiety of substance P, such as substance P-(4-11) or [AcArg6,Sar9,Met(O2)11] substance P-(6-11). Our data therefore indicate that the [3H]protein secretion obtained with substance P results from the activation of NK1 receptors. Moreover, our data suggest that the N-terminal tripeptide of substance P is also active, and could stimulate different phospholipases either by acting through a second functional site on the NK1 receptor or by directly activating G-proteins.

Studies on the cardiovascular effects produced by the spinal action of two substance P analogues in the rat: Evidence for a central catecholaminergic mechanism

The effects of two substance P (SP) analogues, [D-Trp7,9,10]SP (ana1) and [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP-(4-11) (ana2) on mean arterial pressure (MAP) and heart rate (HR) were measured following intrathecal administration at one of three spinal cord levels in rats anaesthetized with sodium pentobarbital. Following an initial increase, a profound and long-lasting fall in MAP and HR occurred when 6.5 nmol of either ana1 or ana2 was injected at T1-T3 or T8-T10. Only transient changes in MAP and a slight increase in HR was observed after injection of either peptide at L2-L4. The profound and long-lasting hypotension and bradycardia induced by ana1 were not significantly altered after intravenous injection of hexamethonium, phentolamine, propranolol, atropine, diphenhydramine, cimetidine, methysergide, naloxone or morphine. However, the biphasic effect of ana1 on MAP was prevented by the intrathecal administration of prazosin and yohimbine, suggesting that a central catecholaminergic mechanism including alpha 1- and alpha 2-adrenergic receptors is involved. The latter treatment did not prevent the tachycardia which occurred when the bradycardia was blocked, indicating that different mechanisms mediate the spinal action of ana1 on MAP and HR. Finally, cervical transection of the spinal cord eliminated the profound and long-lasting depressor effect of ana2, suggesting that a supraspinal mechanism is involved in this cardiovascular response.

Effects of substance P analogues in the rat tail-flick test

Five substance P (SP) analogues which antagonize tachykinins on isolated organs were studied using the tail-flick test. None antagonized the nociceptive effect of 1.9 nmol of physalaemin when studied at the concentrations of 6.5 and 65.0 nmol. [D-alpha Npa7,9,10]SP (65.0 nmol) produced a flaccid paralysis of the hindlimbs and the tail in 2 out of 9 rats; however, [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP-(4-11) (6.5 nmol) produced this type of paralysis in 4 out of 6 rats so treated. The [D-Trp]SP analogue appears to be the most toxic. The results also suggest that the receptor for physalaemin in the spinal cord is different from that in peripheral tissues.

Pharmacological Characterization of Receptor Types

The various biological actions of substance P and related tachykinins/neurokinins are mediated by three different receptor types, which were named NK1, NK2 and NK3 during the Montreal Symposium in 1986 (Henry, 1987). The three receptors have been characterized pharmacologically (Regoli et al., 1987a, Regoli et al., 1988, Regoli et al., 1989), biochemically (Lavielle et al., 1988; Buck et al., 1991), and genetically (Nakanishi, 1991). In vivo activities have been attributed to each receptor by Maggi et al. (1987), Regoli et al. (1988), and Maggi et al. (1993). Cloning and sequencing of receptors and the study of receptor properties of mRNA expressed in special systems (Nakanishi, 1991) appear to validate the three physiological entities.