Maria A. Rivelli

Nociceptin inhibits capsaicin-induced bronchoconstriction in isolated guinea pig lung.

The isolated perfused guinea pig lung was used to investigate the effect of nociceptin against bronchoconstriction elicited by endogenous and exogenous tachykinins. The opioid receptor-like 1 (ORL1) receptor agonist, nociceptin/orphanin FQ (0.001-1 microM) produced a dose-related inhibition of the capsaicin-induced bronchoconstriction (10(-5)-10(3) microg) in isolated guinea pig lung (P<0.05), a response mediated by the release of endogenous tachykinins from lung sensory nerves. The new ORL1 receptor antagonist 1-[(3R, 4R)-1-Cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one (J-113397) (0.3 microM) significantly blocked the inhibitory effect of nociceptin/orphanin FQ (0.01 microM) on capsaicin-induced bronchoconstriction, whereas the non-selective opioid receptor antagonist naloxone (1 microM) had no effect. Nociceptin/orphanin FQ (1 microM) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist neurokinin A. In conclusion, the present data provide evidence that nociceptin inhibits capsaicin-evoked tachykinin release from sensory nerve terminals in guinea pig lung by a prejunctional mechanism. This inhibitory action occurs independently from activation of opioid receptors. The present study also indicates that J-113397 is a potent ORL1 receptor antagonist.

Nociceptin/orphanin FQ inhibits capsaicin-induced guinea-pig airway contraction through an inward-rectifier potassium channel

Nociceptin/orphanin FQ (N/OFQ), an endogenous opioid‐like orphan receptor (NOP receptor, previously termed ORL1 receptor) agonist, has been found to inhibit capsaicin‐induced bronchoconstriction in isolated guinea‐pig lungs and in vivo. The underlying mechanisms are not clear. In the present studies, we tested the effect of N/OFQ on VR1 channel function in isolated guinea‐pig nodose ganglia cells. Capsaicin increased intracellular Ca2+ concentration in these cells through activation of vanilloid receptors. Capsaicin‐induced Ca2+ responses were attenuated by pretreatment of nodose neurons with N/OFQ (1 μM). N/OFQ inhibitory effect on the Ca2+ response in nodose ganglia cells was antagonized by tertiapin (0.5 μM), an inhibitor of inward‐rectifier K+ channels, but not by verapamil, a voltage gated Ca2+ channel blocker, indicating that an inward‐rectifier K+ channel is involved in N/OFQ inhibitory effect. In isolated guinea‐pig bronchus, N/OFQ (1 μM) inhibited capsaicin‐induced airway contraction. Tertiapin (0.5 μM) abolished the N/OFQ inhibition of capsaicin‐induced bronchial contraction. Capsaicin (10 μg) increased pulmonary inflation pressure in the isolated perfused guinea‐pig lungs. This response was significantly attenuated by pretreatment with N/OFQ (1 μM). Tertiapin also abolished the N/OFQ inhibitory effect on capsaicin‐induced bronchoconstriction in perfused lungs. Capsaicin increased the release of substance P and neurokinin A from isolated lungs. N/OFQ (1 μM) blocked the capsaicin‐induced tachykinin release. These results indicate that N/OFQ‐induced hyperpolarization of tachykinin containing airway sensory nerves, through an inward‐rectifier K+ channel activation, accounts for the inhibition of capsaicin‐evoked broncoconstriction.

Pharmacological characterization of postjunctional α-adrenoceptors in human nasal mucosa

Background Functional α1- and α2-adrenoreceptor subtype pharmacology was characterized in an in vitro human nasal mucosa contractile bioassay. Methods Nasal mucosa was obtained from 49 donor patients and mucosal strips were placed in chambers filled with Krebs–Ringer solution and attached to isometric force transducers. Results Nonselective α-adrenoreceptor agonists epinephrine, norepinephrine, and oxymetazoline produced concentration-dependent contractions of isolated human nasal mucosa (pD2= 5.2, 4.9, and 6.5, respectively). The α2-adrenoreceptor agonist BHT-920 (10 μM)–induced contractions were blocked by yohimbine (0.01–1 μM) and prazosin (0.01–1 μM) inhibited the contractile response to the α1-adrenoreceptor agonist phenylephrine (10 μM). Histological analysis showed that phenylephrine and BHT-920 differentially contracted the arteries and veins of human nasal mucosa, respectively. Conclusion Our results indicate that functional α1- and α2-adrenoceptors are present and functional in human nasal mucosa. The a 2-adrenoceptors display a predominant role in contracting the veins and the α1-adrenoceptors appear to preferentially constrict the human nasal arteries.

Novel H3 receptor antagonists. Sulfonamide homologs of histamine

Sulfonamides derived from 4(5)-(omega-aminoalkyl)-1H-imidazoles containing chain lengths of three- to five-carbons were synthesized. Good to moderate H3 receptor binding affinities were observed for several butyl and pentyl homologs, whereas binding affinities were considerably weaker in the propyl series. Separation of the imidazole ring and the sulfonamide unit by a four- or five-carbon tether afforded potent H3 receptor antagonists.

Pharmacological characterization of histamine H3 receptors in human saphenous vein and guinea pig ileum

Studies were performed to assess the functional activity of histamine H3 receptors on neurogenic sympathetic end organ responses in cryopreserved human saphenous vein. (R)-alpha-methylhistamine inhibited electrical field stimulation-evoked contractile responses in a dose dependent manner (pD2 = 8.20). Prazosin (1 microM) and tetrodotoxin (1 microM) blocked the electrical field stimulation-evoked contractile responses in human saphenous vein indicating a sympathetic neural origin of these contractions. The histamine H3 antagonists thioperamide (pA2 = 8.41) and clobenpropit (pA2 = 10.10) produced parallel rightward shifts in the concentration response curve to (R)-alpha-methylhistamine in human saphenous vein and guinea pig ileum (pA2 = 8.59 and 9.83, respectively). Pretreatment with (R)-alpha-methylhistamine (1 microM) did not alter contractions to exogenous norepinephrine in human saphenous vein. In addition, clonidine (pD2 = 10.28) inhibited electrical field stimulation-evoked contractile responses in human saphenous vein which were blocked by yohimbine (30 nM, pA2 = 9.92) but did not alter the (R)-alpha-methylhistamine dose response curve. These results demonstrate the presence of functional presynaptic histamine H3 heteroreceptors on cryopreserved human saphenous vein sympathetic nerves that, upon activation, attenuate electrical field stimulation-evoked contractile responses in this vessel.

Tachykinin NK1 receptor-mediated vasorelaxation in human pulmonary arteries

Tachykinin NK1 receptors are present on human pulmonary arteries. Addition of the specific tachykinin NK1 receptor agonist, [Met-OMe11]substance P produced a concentration-dependent relaxation (0.1 nM to 100 nM) in pulmonary arteries preconstricted with phenylephrine (30 microM). The EC50 (agonist concentration needed to produce 50% of the maximal relaxation) value for [Met-OMe11]substance P was 3.7+/-0.7 nM. The relaxation induced by [Met-OMe11]substance P was selectively inhibited by the tachykinin NK1 receptor antagonist CP 99994 (1 nM), with a pKb of 9.9+/-0.3. Treatment with the tachykinin NK2 receptor antagonist SR 48968 (100 nM) did not significantly affect the vasorelaxation due to [Met-OMe11]substance P (P > 0.05, one-way analysis of variance; ANOVA).

Tryptase-induced airway microvascular leakage in guinea pigs: involvement of tachykinins and leukotrienes

Tryptase, a serine protease synthesized by and stored in mast cells, is implicated as an important mediator in the pathogenesis of airway inflammation. In this study, tryptase was evaluated for its ability to induce microvascular leakage into the airways of guinea pigs. Dose- and time-dependent increases in airway microvascular leakage were produced by intratracheal tryptase (0.3-3 microg). Intratracheal tryptase (3-30 microg) had no effect on airway tone as measured by pulmonary insufflation pressure. Tryptase-induced airway microvascular leakage was partially blocked by the tachykinin NK1 receptor antagonist CP 99994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] and an inhibitor of leukotriene formation SCH 37224 (1-(1,2-dihydro-4-hydroxy-2-oxo-1-phenyl-1,8-naphthyridin-2-yl)pyrrolidinium, hydroxide inner salt). Neither CP 99994 nor SCH 37224 inhibited tryptase proteolytic activity in-vitro. Pretreatment of guinea pigs with histamine H1 receptor antagonists or a tachykinin NK2 receptor antagonist had no affect on the airway microvascular leakage induced by tryptase. It is speculated that tryptase may be important in the pathogenesis of airway inflammation, particularly in disorders that involve increased airway microvascular leakage such as asthma.

Pharmacological Characterization of a Novel α2C-Adrenoceptor Agonist N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1, 4-benzoxazin-6-yl]-N-ethyl-N′-methylurea (Compound A)

We define the pharmacological and pharmacokinetic profiles of a novel α2C-adrenoceptor agonist, compound A [N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1,4-benzoxazin-6-yl]-N-ethyl-N′-methylurea]. This compound has high affinity (Ki) for the human α2C-adrenoceptor (Ki = 12 nM), and 190- to 260-fold selectivity over the α2A- and α2B-adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC50 = 166 nM) and efficacy (Emax = 64%) responses at the α2C-adrenoceptor, much lower potency and efficacy at the α2A-adrenoceptor (EC50 = 1525 nM; Emax = 8%) and α2B-adrenoceptor (EC50 = 5814 nM; Emax = 21%) subtypes, and low or no affinity and functional activity at the α1A-, α1B-, and α1D-adrenoceptor subtypes. In the human saphenous vein postjunctional α2C-adrenoceptor bioassay, compound A functions as a potent agonist (pD2 = 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC50 = 108 nM), and the magnitude of arteriolar contraction reached only 50% of the maximum venular responses. Compound A exhibited no effect on locomotor activity, sedation, and body temperature in mice (up to 100 mg/kg) and did not cause hypertension and mydriasis (30 mg/kg) in conscious rats. Compound A is orally bioavailable (24%) with good plasma exposure. This compound is a substrate for the efflux P-glycoprotein transporter, resulting in very low central nervous system (CNS) penetration. In summary, compound A is a highly selective, orally active, and non-CNS-penetrating α2C-adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.

Bronchoconstrictor effect of the tachykinin NK3-receptor agonists [MePhe7]-neurokinin B and senktide in the isolated guinea pig lung

ABSTRACT To determine whether bronchoconstriction can be mediated via the tachykinin NK3 receptors, isolated guinea pig lungs were challenged with the exogenous tachykinin NK3-receptor agonists [MePhe7]-neurokinin B ([MePhe7]-NKB) and senktide. [MePhe7]-NKB induced bronchoconstriction (EC50 = 11.8 ± 1.7 μM) that was significantly inhibited by the tachykinin NK3-receptor antagonist SB 223412 at 10 μM (EC50 = 24.4 ± 4.5 μM). Senktide also induced bronchoconstriction (EC50 = 96.2 ± 20.3 μM) and the bronchoconstriction was significantly reduced by SB 223412 at 1 and 10 μM (EC50 = 270.8 ± 78.9 μM and 388.3 ± 105.5 μM, respectively). Although the authors demonstrated that SB 223412, [MePhe7]-NKB, and senktide are potent and selective for the tachykinin NK3 receptors in binding and functional (Ca2+ mobilization) assays, the tachykinin NK1-receptor antagonist CP 99,994 at 1 μM (EC50 = 32.7 ± 8.5 μM) produced inhibition of [MePhe7]-NKB–induced bronchoconstriction, whereas the tachykinin NK2-receptor antagonist SR 48968 at 0.1 μM (EC50 = 213.2 ± 42.9 μM) blocked senktide-induced bronchoconstriction. These data suggest that [MePhe7]-NKB and senktide caused bronchoconstriction in guinea pig through activation of the tachykinin NK3-receptors but the tachykinin NK1- and/or NK2-receptors are also involved in the response.

Effects of an α2-Adrenoceptor Agonist in Nasal Mucosa

arteries and arterioles (resistance vessels), abundant arteriovenous anastomoses, and a subepithelial and periglandular capillary network (exchange vessels), which drain into collecting veins and large venous sinusoids (capacitance vessels). Although the nasal vasculature is under nervous and reflex control receiving innervation from sympathetic, parasympathetic and non-myelinated sensory nerves, the dominant motor supply is sympathetic. When stimulated, the sympathetic nerves release postganglionic transmitters, such as norepinephrine, that bind to a1and a2-adrenoceptors in the vasculature resulting in vasoconstriction. Nasal congestion, one of the symptoms of rhinitis, is induced by an increased blood flow leading to the filling of venous sinusoids with vascular congestion, swelling of the inferior turbinates and obstruction of nasal airflow. Adrenoceptor-agonists such as the selective a1-adrenoceptor agonist phenylephrine and the non-selective a-adrenoceptor agonist oxymetazoline constrict vessels and decrease the blood flow in the nasal mucosa. However, these agonists also have the potential to increase significantly the blood pressure leading to cardiovascular liabilities such as hypertension and stroke. In the present study, we evaluate the effects of the selective a2-adrenoceptor agonist BHT-920 in different in vitro nasal mucosal contractile bioassays [1], and we characterize the decongestant activity profile of the BHT-920 in a model of nasal congestion using acoustic rhinometry [2]. Nasal mucosae were removed from dog, pig and human and mucosal strips were placed in organ chambers filled with Krebs buffer, maintained at 37°C and aerated with 95% O2 – 5% CO2, and attached to force transducers for recording of isometric tension. Both a1and a2-adrenoceptor agonists dose-dependently contracted nasal mucosa in these different species indicating that a1and a2-adrenoceptors are present in nasal mucosae and modulate nasal mucosa vascular tone. Histological studies were performed in pig to identify the contribution of arteries and veins to the a1and a2adrenoceptor-mediated contractions. Tissues in organ baths were first challenged with 10 M phenylephrine or 10 M BHT-920. Then tissues were cut from the baths at the contraction peak and put into formalin. Formalin-fixed tissues were processed and paraffin embedded. Paraffin embedded tissues were sliced to 5mm and stained with H & E stain. We found that BHT-920 preferentially contracted the veins while phenylephrine induced contraction in both arteries and veins. In another set of experiments, pig nasal mucosa were embedded in low melt agarose, then cut into slices placed in tissue culture dish and incubated at 37°C overnight with 95% O2 – 5% CO2. Tissues were challenged with the non-selective aadrenoceptor agonist epinephrine, phenylephrine or BHT920. Tissue image was recorded and vascular size area analysis performed with Image J Software. Real time vessel contraction was evaluated as a percent of area decrease. We found that a2-adrenoceptor agonist preferentially contract veins while a1-adrenoceptor-agonist appears to preferentially act on the arteries of pig nasal mucosa. Histology study also demonstrated that sinusoids in pig nasal mucosa comprise the largest volume of blood. The decongestant activities of the a1-adrenoceptor agonist phenylephrine and the a2-adrenoceptor agonist BHT-920 against nasal congestion were evaluated by using the acoustic rhinometry technique in cat. Non-invasive measurements of nasal cross-sectional area and nasal volume made were performed. Area-distance curves provided estimates of crossSHORT COMMUNICATION