The positive inotropic and chronotropic effects of evodiamine and rutaecarpine, indoloquinazoline alkaloids isolated from the fruits of Evodia rutaecarpa, on the guinea-pig isolated right atria: possible involvement of vanilloid receptors

Cardiotonic effects of evodiamine and rutaecarpine, constituents of the fruits of Evodia rutaecarpa Bentham Rutaceae, were evaluated on guinea pig isolated atria. Comparison with capsaicin, a vanilloid receptor agonist, revealed similar positive inotropic and chronotropic activity, as judged from antagonistic effects of the competitive vanilloid receptor (capsaicin receptor) antagonist capsazepine, the non-competitive vanilloid receptor antagonist ruthenium red, the calcitonin gene related peptide antagonist CGRP(8-37), the P2X purinoceptor antagonist PPADS, and various desensitization studies. Evodiamine and rutaecarpine produced transient positive inotropic and chronotropic effects on the guinea-pig isolated atria, followed by a desensitizing effect to additional administration. Dose-response relationships for evodiamine, rutaecarpine and capsaicin were obtained. All the compounds evoked positive inotropic and chronotropic effects in a concentration-dependent manner. Maximal contractions for evodiamine, rutaecarpine and capsaicin were observed at concentrations of 1 microM, 3 microM and 0.3 microM, respectively. The cardiotonic responses evoked by both evodiamine and rutaecarpine were shifted to the right by capsazepine, an established antagonist of vanilloid receptor (capsaicin-receptor). The effects of both evodiamine (1 microM) and rutaecarpine (3 microM) were abolished by pretreatment with a desensitizing dosage of capsaicin (1 microM), developing cross-tachyphylaxis between these compounds. The effects of evodiamine (1 microM), rutaecarpine (3 microM) and capsaicin (0.3 microM) were also significantly reduced by pretreatment with ruthenium red (10 microM) and CGRP (8-37) (10 microM). The effects of evodiamine, rutaecarpine and capsaicin were not affected by pretreatment with PPADS (100 microM), a highly selective P2X purinoceptor antagonist, and the possibility of the involvement of the P2X purinoceptor was excluded. These results suggest that the positive inotropic and chronotropic effects on the guinea-pig isolated right atria induced by both evodiamine and rutaecarpine could be attributed to their interaction with vanilloid receptors and the resultant release of CGRP, a cardiotonic neurotransmitter, from capsaicin-sensitive nerves as with capsaicin.     

Differential effects of intraplantar capsazepine and ruthenium red on capsaicin-induced desensitization in mice

Intraplantar injection of capsaicin (1.6 microg/paw) into the mouse hindpaw produced an acute paw-licking/biting response. This study was designed (1) to investigate the antinociceptive effects of intraplantar administration of capsazepine, a competitive vanilloid receptor antagonist, and ruthenium red, a noncompetitive antagonist, in the nociceptive licking/biting response induced by intraplantar injection of capsaicin, and (2) to determine whether these compounds were able to prevent capsaicin-induced desensitization in mice. Both capsazepine and ruthenium red produced a dose-dependent reduction in the capsaicin-induced nociceptive response. In licking/biting response to intraplantar capsaicin, ruthenium red was more potent than capsazepine in producing antinociceptive activity as assayed by the capsaicin test. The first injection of capsaicin induced a profound desensitization to the second and third injections of capsaicin at the interval of 15 or 30 min. The capsaicin-induced desensitization was prevented dose-dependently by antinociceptive doses of capsazepine, whereas ruthenium red in doses exhibiting antinociceptive activity was without effect on capsaicin-induced desensitization. The present results suggest that both capsazepine and ruthenium red can produce a local peripheral antinociceptive action, which may be mediated by inhibiting the membrane ion channel activated by capsaicin. In addition, these data suggest that capsazepine may act in the mechanism clearly different from ruthenium red in the capsaicin-induced nociceptive desensitization.     

Pharmacological Characterization of the Vanilloid Receptor in the Rat Isolated Vas Deferens

The present study set out to further characterize the vanilloid receptor in the rat isolated vas deferens. In this preparation, both capsaicin and resiniferatoxin (RTX) evoked a concentration-dependent inhibition in the amplitude of electrically-evoked contractions with pEC50 values of 7.62 ± 0.03 and 12.2 ± 0.21 respectively. Responses to capsaicin were fast in onset and faded rapidly over a 30-min exposure period, whereas those to RTX were slow in onset and well maintained, an observation believed to reflect pharmacokinetic differences in the rate of penetration to the vanilloid receptor. Responses to both agonists showed mutual cross-desensitization and were antagonized by both the vanilloid-receptor antagonist capsazepine and the ion-channel blocker ruthenium red. The capsaicin analogue, olvanil failed to either mimic or antagonize capsaicin-evoked responses in the rat isolated vas deferens, an effect at variance with previous observations in other tissues. The reason for these differences is unclear, but the possibility of multiple classes of receptor cannot at this stage be ruled out.     

Vanilloid receptors on capsaicin-sensitive sensory nerves mediate relaxation to methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arteries

In the present study, the vasodilator actions of methanandamide and capsaicin in the rat isolated mesenteric arterial bed and small mesenteric arterial segments were investigated. Methanandamide elicited concentration-dependent relaxations of preconstricted mesenteric arterial beds (pEC(50)=6.0+/-0.1, E(max)=87+/-3%) and arterial segments (pEC(50)=6.4+/-0.1, E(max)=93+/-3%). In arterial beds, in vitro capsaicin pre-treatment blocked vasorelaxation to 1 and 3 microM methanandamide, and reduced to 12+/-7% vasorelaxation to 10 microM methanandamide. Methanandamide failed to relax arterial segments pre-treated in vitro with capsaicin. In arterial beds from rats treated as neonates with capsaicin to cause destruction of primary afferent nerves, methanandamide at 1 and 3 microM did not evoke vasorelaxation, and relaxation at 10 microM methanandamide was reduced to 26+/-4%. Ruthenium red (0.1 microM), an inhibitor of vanilloid responses, attenuated vasorelaxation to methanandamide in arterial beds (pEC(50)=5.6+/-0.1, E(max)=89+/-1%). Ruthenium red at 1 microM abolished the response to 1 microM methanandamide, and greatly attenuated relaxation at 3 and 10 microM methanandamide in arterial beds. In arterial segments, ruthenium red (0.15 microM) blocked vasorelaxation to methanandamide, but not to CGRP. In arterial segments, the vanilloid receptor antagonist capsazepine (1 microM) inhibited, and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8 - 37) (3 microM) abolished, methanandamide-induced relaxations. CGRP(8 - 37), but not capsazepine, attenuated significantly relaxation to exogenous CGRP. These data show that capsaicin and ruthenium red attenuate vasorelaxation to methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arterial segments. In addition, CGRP(8 - 37) and capsazepine antagonize responses to methanandamide in mesenteric arterial segments. In conclusion, vanilloid receptors on capsaicin-sensitive sensory nerves play an important role in the vasorelaxant action of methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arterial segments.     

Capsazepine as a selective antagonist of capsaicin-induced activation of C-fibres in guinea-pig bronchi.

We investigated the action of capsazepine, an antagonist of the actions of capsaicin on sensory neurones, on the contractile responses evoked by capsaicin or by electrical field stimulation (EFS) in guinea-pig bronchi. Capsazepine (10(-5) M) selectively inhibited responses to capsaicin, producing a significant change in EC50 values but not the Hill coefficient (nH), suggesting that capsazepine acts as a competitive antagonist (apparent pKB = 5.12) whereas ruthenium red is a non-competitive antagonist. Capsazepine and ruthenium red were without effect on EFS-induced responses.     

Effects of ruthenium red and capsazepine on C-fibres in the rabbit iris.

1. We have investigated the effects of ruthenium red and capsazepine on a C-fibre-smooth muscle preparation (the rabbit isolated iris sphincter muscle). 2. Like capsaicin, ruthenium red and capsazepine were found to produce contractions in a concentration-dependent manner. C-fibre activation was held to be responsible since the contractions could be inhibited by tachykinin receptor blockade. 3. Both ruthenium red and capsazepine inhibited capsaicin-induced contractions concentration-dependently; the pIC50 values were 5.1 and 4.9, respectively. The contractions induced by bradykinin, which, like capsaicin, acts by releasing tachykinins from C-fibres, were also inhibited by ruthenium red and capsazepine in a concentration-dependent manner; the pIC50 values were 4.1 and 4.6, respectively. 4. Electrically evoked, tachykinin-mediated contractions were inhibited by ruthenium red and capsazepine in a concentration-dependent manner; the pIC50 values were 4.3 and 4.5, respectively. 5. The contractile response to neurokinin A (NKA) was inhibited by capsazepine (and by capsaicin), but not by ruthenium red, in a concentration-dependent manner; the pIC50 value was 4.3. 6. The results suggest that, besides their ability to antagonize capsaicin, ruthenium red and capsazepine possess a weak capsaicin-like effect. Conceivably, capsazepine interacts with binding sites for capsaicin, acting as a partial agonist/antagonist, while ruthenium red interacts with capsaicin-operated cation channels. The inhibition of electrically evoked- or bradykinin-induced responses by capsazepine and ruthenium red suggests that capsaicin/capsazepine binding sites and capsaicin-operated cation channels play a role in the process of transmitter release in response not only to capsaicin but also to other C-fibre stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens

1. This study was directed at exploring the structure-activity relationship for anandamide and certain of its analogues at the rat VR1 receptor in transfected cells and at investigating the relative extent to which anandamide interacts with CB(1) and vanilloid receptors in the mouse vas deferens. 2. pK(i) values for displacement of [(3)H]-resiniferatoxin from membranes of rVR1 transfected CHO cells were significantly less for anandamide (5.78) than for its structural analogues N-(4-hydroxyphenyl)-arachidonylamide (AM404; 6.18) and N-(3-methoxy-4-hydroxy)benzyl-arachidonylamide (arvanil; 6.77). 3. pEC(50) values for stimulating (45)Ca(2+) uptake into rVR1 transfected CHO cells were significantly less for anandamide (5.80) than for AM404 (6.32) or arvanil (9.29). Arvanil was also significantly more potent than capsaicin (pEC(50)=7.37), a compound with the same substituted benzyl polar head group as arvanil. 4. In the mouse vas deferens, resiniferatoxin was 218 times more potent than capsaicin as an inhibitor of electrically-evoked contractions. Both drugs were antagonized to a similar extent by capsazepine (pK(B)=6.93 and 7.18 respectively) but were not antagonized by SR141716A (1 microM). Anandamide was less susceptible than capsaicin to antagonism by capsazepine (pK(B)=6.02) and less susceptible to antagonism by SR141716A (pK(B)=8.66) than methanandamide (pK(B)=9.56). WIN55212 was antagonized by SR141716A (pK(B)=9.02) but not by capsazepine (10 microM). 5. In conclusion, anandamide and certain of its analogues have affinity and efficacy at the rat VR1 receptor. In the mouse vas deferens, which seems to express vanilloid and CB(1) receptors, both receptor types appear to contribute to anandamide-induced inhibition of evoked contractions.     

Pharmacological investigation of hydrogen sulfide (H2S) contractile activity in rat detrusor muscle

We have investigated the mechanism through which hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat isolated urinary bladder. Sodium hydrogen sulfide (NaHS), a donor of H2S, produced concentration-dependent contractile responses (pEC50=3.5+/-0.1) that were unaffected by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine (30 microM) and SB 366791 (10 microM) and by the N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CTX; 100 nM). In contrast, the unselective transient receptor potential (TRP) cation channels blocker ruthenium red (30 microM) almost abolished NaHS-induced contractions. Ruthenium red (30 microM) greatly reduced capsaicin-induced contractions, whereas it did not attenuate the contractile response to neurokinin A. The putative TRPV1 receptor antagonist iodo-resiniferatoxin, from 100 nM upward, produced agonist responses per se, and could not be tested against NaHS. We conclude that H2S either acts at TRPV1 receptorial sites unblocked by capsazepine or SB 366791, or stimulates a still unidentified transient receptor potential-like channel co-expressed with TRPV1 on sensory neurons.     

Lafutidine facilitates calcitonin gene-related peptide (CGRP) nerve-mediated vasodilation via vanilloid-1 receptors in rat mesenteric resistance arteries

Lafutidine is a histamine H(2)-receptor antagonist with gastric antisecretory and gastroprotective activity associated with activation of capsaicin-sensitive nerves. The present study examined the effect of lafutidine on neurotransmission of capsaicin-sensitive calcitonin gene-related peptide (CGRP)-containing vasodilator nerves (CGRPergic nerves) in rat mesenteric resistance arteries. Rat mesenteric vascular beds were perfused with Krebs solution and vascular endothelium was removed by 30-s perfusion with sodium deoxycholate. In preparations preconstricted by continuous perfusion of methoxamine (alpha(1) adrenoceptor agonist), perfusion of lafutidine (0.1 - 10 microM) concentration-dependently augmented vasodilation induced by the periarterial nerve stimulation (PNS, 1 Hz) without affecting vasodilation induced by exogenous CGRP (10 pmol) injection. Perfusion of famotidine (H(2)-receptor antagonist, 1 - 100 microM) had no effect on either PNS-induced or CGRP-induced vasodilation. Perfusion of lafutidine concentration-dependently augmented vasodilation induced by a bolus injection of capsaicin (vanilloid-1 receptor agonist, 30 pmol). The presence of a vanilloid-1 receptor antagonist, ruthenium red (10 microM) or capsazepine (5 microM), abolished capsaicin-induced vasodilation and significantly decreased the PNS-induced vasodilation. The decreased PNS-induced vasodilation by ruthenium red or capsazepine was not affected by perfusion of lafutidine. These results suggest that lafutidine facilitates CGRP nerve-mediated vasodilation by modulating the function of presynaptic vanilloid-1 receptors located in CGRPergic nerves.     

Lactic Acid-Induced Plasma Protein Extravasation in Rat Airways by Stimulation of Sensory Nerves and NK1 Receptor Activation

Abstract: Locally applied lactic acid and capsaicin caused extravasation of Evans blue dye in trachea, main bronchi and nasal mucosa of anaesthetized rats. In animals pretreated with capsaicin to deplete sensory neuropeptides, the lactic acid response was abolished in main bronchi and highly reduced in trachea. Pretreatment with the NK1 receptor antagonist, RP 67580 (3 mgxkg^?1 intravenously), markedly inhibited the lactic acid-induced extravasation at all levels; similar pretreatment with NK2 receptor antagonist, SR 48968 (0.5 mgxkg^?1 intravenously), was ineffective. Locally applied ruthenium red (a transmembrane Ca²⁺ fluxes inhibitor), capsazepine (a capsacin receptor antagonist) and diclofenac intraperitoneally (a cyclooxygenase blocker) did not change the lactic acid effect, while the capsaicin response was only diminished in bronchi by local pretreatment with ruthenium red. In conclusion locally applied lactic acid in rat trachea and nasal cavity activated capsaicin sensitive sensory nerve endings producing plasma protein extravasation. This reaction was shown to be mediated by tachykinins acting on the NK1 receptor through a mechanism which appeared to be resistant to capsazepine and ruthenium red and independent of cyclooxygenase products. In comparison the effect of capsacin was partially ruthenium red-sensitive but not influenced by capsazepine.     

Synthesis and characterization of a selective peptide antagonist of neuropeptide Y vascular postsynaptic receptors.

1. A cyclic dimeric nonapeptide neuropeptide Y (NPY) receptor antagonist, 1229U91, was synthesized by Fmoc chemistry and dimerised in solution. Its effects were assayed in mesenteric arteries from rats and mice, and in rat vas deferens. 2. Mesenteric arteries were cannulated and pressurised to 55 mmHg and the external diameters continuously measured. NPY, PYY, Leu31Pro34NPY and NPY(13-36) each caused concentration-related contractions with the order of potency PYY > or = Leu31Pro34NPY = NPY > NPY (13-36), consistent with the Y1 receptor subtype. 3. 1229U91 had no agonist activity in the arteries but caused a concentration-related rightward shift of NPY (mouse arteries) or Leu31Pro34NPY (rat) concentration-response curves. The antagonism was competitive with pKBS of 7.69 +/- 0.15 and 7.47 +/- 0.13 in the mouse and rat arteries, respectively. 4. Sympathetic nerves in the vas deferens were stimulated with a single electrical field pulse every 20 s and the twitch responses recorded. NPY, PYY, Leu31Pro34NPY and NPY(13-36) inhibited the twitches with the order of potency PYY > NPY > NPY(13-36) >> Leu31Pro34NPY, consistent with the Y2 receptor subtype. 5. 1229U91 inhibited the vas deferens twitch with a shallow concentration-response curve and a time-course of inhibition distinct from that of NPY. 1229U91 (30 microM) did not cause a rightward shift of the NPY concentration-response curve. 1229U91 is at least 5 orders of magnitude less potent in the vas deferens than in rat brain Y2 binding assays reported by others, suggesting that the brain and vas deferens Y2 receptors are different. 6. It is concluded that 1229U91 is a competitive antagonist of NPY Y1 vascular receptors and has additional properties that inhibit the electrically evoked twitch of the rat vas deferens.     

Ruthenium red, but not capsazepine reduces plasma extravasation by cigarette smoke in rat airways.

1. Cigarette smoke increases vascular permeability in rat airways by activating release of tachykinin from capsaicin-sensitive sensory nerves. However, the mechanism by which cigarette smoke induces secretion of sensory neuropeptides is unknown. Here we hypothesized that cigarette smoke activates sensory nerve endings via a mechanism similar to that of capsaicin. 2. We studied the effects of ruthenium red, an inorganic dye which blocks the cation influx promoted by capsaicin and of the capsaicin antagonist capsazepine on the increase in vascular permeability produced by cigarette smoke, capsaicin, hypertonic saline and substance P in the trachea of pentobarbitone anaesthetized rats. We also investigated the ability of cigarette smoke to desensitize sensory nerve fibres. 3. Ruthenium red (10 mM) by aerosol blocked the increase in vascular permeability induced by capsaicin (0.5 microM) and reduced the response to cigarette smoke (5 puffs) but did not affect responses evoked by hypertonic saline (7.2%) or by substance P (10 microM) (all given by aerosol). Aerosols of capsazepine (0.1 mM) prevented extravasation by capsaicin, but did not inhibit response to cigarette smoke, hypertonic saline or substance P. Finally, pre-exposure to a high dose of cigarette smoke (10 puffs) prevented the extravasation caused by cigarette smoke (5 puffs) itself and by intravenous capsaicin (150 micrograms kg-1), but not that by intravenous substance P (10 nmol kg-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

A pharmacological study with substance K: evidence for multiple types of tachykinin receptors

The substance P antagonist [D-Arg,D-Pro,D-Trp,Leu]SP (RPTTL-SP) produced a simple competitive antagonism of the responses to substance K (SK) and substance P (SP) in a variety of tissues. In the guinea-pig ileum and field stimulated rat vas deferens the pKB values against SK were similar to those against SP indicating a single population of receptors. In the guinea-pig urinary bladder the pKB value against SK was significantly higher suggesting the existence of at least two subtypes of tachykinin receptor in this tissue. In the hamster urinary bladder no antagonism was observed with up to 30 microM RPTTL-SP indicating the possibility of a third type of receptor.     

Differential effects of suramin on P2-purinoceptors mediating contraction of the guinea-pig vas deferens and urinary bladder.

1. The effect of the P2-purinoceptor antagonist, suramin, was investigated on contractions of the guinea-pig vas deferens and urinary bladder induced by adenosine 5'-triphosphate (ATP) and by the other naturally occurring nucleoside triphosphates. 2. ATP, guanosine 5'-triphosphate (GTP), cytidine 5'-triphosphate (CTP), inosine 5'-triphosphate (ITP) and uridine 5'-triphosphate (UTP) (0.1-500 microM) each contracted both the guinea-pig bladder and the guinea-pig vas deferens. In the vas deferens the order of potency of the nucleotides was ATP >> CTP > GTP > or = UTP = ITP, and in the bladder it was ATP >> CTP = GTP > UTP = ITP, although maximal responses to these agonists were not achieved in either tissue. 3. Suramin (30 microM-1 mM) dose-dependently inhibited ATP-induced contractions of the bladder in an apparently non-competitive manner, causing a reduction in the slope of the concentration-response curve to ATP. In contrast, suramin (5 microM-1 mM) had little inhibitory effect on ATP-induced contractions of the vas deferens, and indeed at concentrations of 100 microM and above markedly potentiated high concentrations of ATP (100-500 microM). The contractions induced by CTP, GTP, UTP and ITP (1-500 microM) were, however, abolished by suramin (1 mM) in each tissue. 4. Desensitization of the P2X purinoceptors in the guinea-pig vas deferens with adenosine 5'-alpha,beta-methylenetriphosphonate (AMPCPP) (300 microM) abolished contractions induced by ATP (1 microM-1 mM) in the absence of suramin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of piperine, the pungent component of black pepper, at the human vanilloid receptor (TRPV1)

1. We have characterised the effects of piperine, a pungent alkaloid found in black pepper, on the human vanilloid receptor TRPV1 using whole-cell patch-clamp electrophysiology. 2. Piperine produced a clear agonist activity at the human TRPV1 receptor yielding rapidly activating whole-cell currents that were antagonised by the competitive TRPV1 antagonist capsazepine and the non-competitive TRPV1 blocker ruthenium red. 3. The current-voltage relationship of piperine-activated currents showed pronounced outward rectification (25+/-4-fold between -70 and +70 mV) and a reversal potential of 0.0+/-0.4 mV, which was indistinguishable from that of the prototypical TRPV1 agonist capsaicin. 4. Although piperine was a less potent agonist (EC50=37.9+/-1.9 microM) than capsaicin (EC50=0.29+/-0.05 microM), it demonstrated a much greater efficacy (approximately two-fold) at TRPV1. 5. This difference in efficacy did not appear to be related to the proton-mediated regulation of the receptor since a similar degree of potentiation was observed for responses evoked by piperine (230+/-20%, n=11) or capsaicin (284+/-32%, n=8) upon acidification to pH 6.5. 6. The effects of piperine upon receptor desensitisation were also unable to explain this effect since piperine resulted in more pronounced macroscopic desensitisation (t(1/2)=9.9+/-0.7 s) than capsaicin (t(1/2)>20 s) and also caused greater tachyphylaxis in response to repetitive agonist applications. 7. Overall, our data suggest that the effects of piperine at human TRPV1 are similar to those of capsaicin except for its propensity to induce greater receptor desensitisation and, rather remarkably, exhibit a greater efficacy than capsaicin itself. These results may provide insight into the TRPV1-mediated effects of piperine on gastrointestinal function.     

Involvement of multiple receptors in the biological effects of calcitonin gene-related peptide and amylin in rat and guinea-pig preparations.

1. The activity of rat alpha and beta calcitonin gene-related peptide (CGRP) as compared to the structurally related peptide, rat amylin, has been investigated in the guinea-pig isolated left atrium (electrically driven), in mucosa-free strips from the base of the guinea-pig urinary bladder and in the rat isolated vas deferens (pars prostatica). The antagonist activity of the C-terminal fragment of human alpha CGRP, alpha CGRP(8-37), was also investigated. 2. In the guinea-pig isolated left atrium the three peptides produced a concentration-related positive inotropic effect, amylin being about 16 and 31 times less potent than alpha or beta CGRP, respectively. Human alpha CGRP(8-37) produced a rightward displacement of the log concentration-response curve to the three agonists tested, without depression of maximal response attainable. Apparent pKB values calculated on the basis of the displacement produced by 1 microM human alpha CGRP(8-37) indicated an agonist-independent affinity of the antagonist (6.66 +/- 0.11 for alpha CGRP, 6.42 +/- 0.17 for beta CGRP and 6.95 +/- 0.11 for amylin). 3. In the guinea-pig isolated urinary bladder, alpha or beta CGRP or amylin produce a concentration-related inhibition of twitch contractions evoked by train electrical field stimulation (10 Hz frequency, 0.25 ms duration at 100 V for 0.5 s every 60 s). Amylin was about 100 times less potent than alpha or beta CGRP. Human alpha CGRP(8-37) (3 microM) did not significantly affect the inhibitory action of the three agonists tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder

TRPA1 is a member of the transient receptor potential (TRP) channel family present in sensory neurons. Here we show that vanilloid receptor (TRPV1) stimulation with capsaicin and activation of TRPA1 with allyl isothiocyanate or cinnamaldehyde cause a graded contraction of the rat urinary bladder in vitro. Repeated applications of maximal concentrations of the agonists produce desensitization to their contractile effects. Moreover, contraction caused by TRPA1 agonists generates cross-desensitization with capsaicin. The TRP receptor antagonist ruthenium red (10-100 microM) inhibits capsaicin (0.03 microM), allyl isothiocyanate (100 microM) and cinnamaldehyde (300 microM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10 microM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10-1000 microM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [(3)H]-resiniferatoxin. Allyl isothiocyanate or cinnamaldehyde-mediated contractions of rat urinary bladder, which rely on external Ca(2+) influx, are significantly inhibited by tachykinin receptor antagonists as well as by tetrodotoxin (1 microM) or indomethacin (1 microM). Allyl isothiocyanate-induced contraction is not changed by atropine (1 microM) or suramin (300 microM). The exposure of urinary bladders to allyl isothiocyanate (100 microM) causes an increase in the prostaglandin E(2) and substance P levels. Taken together, these results indicate that TRPA1 agonists contract rat urinary bladder through sensory fibre stimulation, depending on extracellular Ca(2+) influx and release of tachykinins and cyclooxygenase metabolites, probably prostaglandin E(2). Thus, TRPA1 appears to exert an important role in urinary bladder function.     

Suramin: a selective inhibitor of purinergic neurotransmission in the rat isolated vas deferens.

The effects of the putative P2 purinoceptor antagonist suramin on contractile responses of the rat isolated vas deferens to electrical field stimulation and exogenously applied drugs (alpha,beta-methylene ATP and noradrenaline) were investigated. Suramin was devoid of agonist activity in the rat vas deferens. The response of the vas deferens to single pulse field stimulation was characteristically biphasic with a large first component peaking between 260-300 ms after the stimulus followed by a second smaller component peaking at about 650 ms post-stimulus. Suramin (100 nM-1 mM) selectively impaired the first, purinergic phase of the response to single pulse field stimulation but was without effect on the second, noradrenergic component. The response of the vas deferens to trains of electrical field stimuli (10 Hz for 10 s) was also biphasic. Suramin (1 microM-1 mM) reduced the first (less than 1 s) phase of the response by 30%, the second (greater than 5 s) plateau phase by 50% and inhibited the intermediate (2-4 s) phase by 80%. Dose-contact periods of 20 or 30 min respectively were sufficient to achieve equilibration of the inhibitory effects of suramin against the responses to single pulse field stimulation or trains of pulses. Following 30 min incubation with 1 mM suramin, the remaining first and second phase components of the response to trains of pulses were impaired and subsequently abolished by the alpha 1-adrenoceptor antagonist WB4101 establishing their noradrenergic origin. Suramin (300 microM) abolished responses of the vas deferens to alpha,beta-methylene ATP but was without effect on those to noradrenaline. Suramin (30 microM) induced a rightward shift in the concentration-response relationship to alpha,beta-methylene ATP that was associated with a significant 40% increase in the maximum response, but did not modify responses to noradrenaline. The inhibitory effects of suramin (3-300 microM) on responses of the vas deferens to approximate EC50 concentrations of alpha,beta-methylene ATP were reversible on repeated washout for 40-60 min. These results reveal suramin to be a useful pharmacological tool for the study of purinergic neurotransmission in rodent vasa deferentia.     

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.     

Differential blocking actions of idazoxan against the inhibitory effects of 6-fluoronoradrenaline and clonidine in the rat vas deferens

The prejunctional inhibitory effects of clonidine and 6-fluoronoradrenaline (6-FNA) have been evaluated in the isolated prostatic segment of the rat vas deferens, against the twitch response evoked by low frequency (0.1 Hz) field stimulation. The inhibitory potency of 6FNA was significantly increased in the presence of cocaine (1 microM) or pargyline (10 microM), but was not modified in the vas deferens from rats pretreated with reserpine when the endogenous levels of noradrenaline (NA) were decreased by 97%. Clonidine was significantly more potent than 6-FNA as an inhibitory agonist, and the potency of clonidine was not modified after cocaine, pargyline or reserpine. The alpha 2-adrenoceptor blocking agent idazoxan, was a competitive antagonist against the inhibitory effects of clonidine under all experimental conditions. In contrast, the only antagonism shown by idazoxan against the inhibitory effects of 6-FNA was in the presence of cocaine (1 microM), and this antagonist effect of idazoxan was not concentration-related. Low concentrations of 6-FNA caused concentration-dependent facilitatory effects on the twitch response, which were significantly greater after treatment with idazoxan (1 microM) in reserpine-treated vas deferens. These facilitatory effects of 6-FNA were always observed in the presence of prazosin (300 nM) and also after treatment of the preparations with phenoxybenzamine (10 microM), a concentration which abolished the inhibitory actions of both clonidine and 6-FNA. The facilitatory effects on the twitch response induced by low concentrations of 6-FNA are therefore unlikely to be due to either alpha 1- or alpha 2-adrenoceptor stimulation. In conclusion, the failure of idazoxan to block the inhibitory effects of 6-FNA, while exerting a potent competitive antagonism of clonidine-induced inhibitory effects, supports the proposal that alpha 2-adrenoceptors may in fact be subdivided into two subclasses, involving imidazoline and phenylethylamine recognition sites.