In vitro and in vivo studies of dansylated compounds, the putative agonists and antagonists on neuropeptide FF receptors

To further evaluate the importance of C-terminal modification of neuropeptide FF (NPFF), in the present work, four dansylated NPFF analogues, including two putative agonists (dansyl-PQRFamide and dansyl-GSRFamide) and two putative antagonists (dansyl-PQRamide and dansyl-GSRamide), were synthesized and investigated to address their potencies and efficacies in a series of in vitro and in vivo assays. (1) In the isolated mouse colon bioassay, the four dansylated compounds showed agonistic profiles: both dansyl-GSRFamide (1-10 microM) and dansyl-GSRamide (1-10 microM) dose-dependently caused colonic contractions, which were attenuated by pretreatment with BIBP3226; dansyl-PQRFamide and dansyl-PQRamide evoked modest colonic contractions at a high dose of 50 microM. (2) In urethane-anaesthetized rats, both dansyl-PQRFamide (50-300 nmol/kg, i.v.) and dansyl-GSRFamide (15-50 nmol/kg, i.v.) dose-dependently increased the mean arterial pressure and heart rate in a manner similar to NPFF (50-300 nmol/kg, i.v.); on the contrary, the two putative antagonists (100-800 nmol/kg, i.v.) decreased blood pressure in a dose-dependent manner. All the results suggest that dansyl-PQRFamide and dansyl-GSRFamide are NPFF full agonists; in contrast, dansyl-GSRamide and dansyl-PQRamide behave as agonists in vitro and antagonists in vivo on NPFF receptors. The findings reveal that the C-terminal Phe might be a crucial residue to determine the efficacy. In addition, the novel analogue dansyl-GSRFamide may be developed as a highly potent agonist to investigate the NPFF system.     

In vivo inhibition of neuropeptide FF agonism by BIBP3226, an NPY Y1 receptor antagonist

BIBP3226 {(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide} was recently shown to display relatively high affinities for neuropeptide FF (NPFF) receptors and exhibit antagonist activities towards NPFF receptors in vitro. The present study was undertaken to investigate the antagonistic effects of BIBP3226 on several in vivo pharmacologic profiles induced by exogenous NPFF and NPVF. (1) BIBP3226 (5 nmol) injected into the third ventricle completely antagonized the hypothermic effects of NPFF (30 nmol) and NPVF (30 nmol) after cerebral administration in mice; (2) BIBP3226 (5 nmol, i.c.v.) prevented the anti-morphine actions of NPFF (10 nmol, i.c.v.) in the mouse tail-flick assay; (3) in urethane-anaesthetized rats, both NPFF (200 nmol/kg, i.v.) and NPVF (200 nmol/kg, i.v.) increased the mean arterial blood pressure, which were significantly reduced by pretreatment with BIBP3226 (500 nmol/kg, i.v.). Collectively, these data suggest that BIBP3226, a mixed antagonist of NPY Y1 and NPFF receptors, shows in vivo antagonistic effects on NPFF receptors. In addition, it seems to be clear that the in vivo pharmacological profiles of NPFF are mediated directly by NPFF receptors.     

Identification and characterization of novel mammalian neuropeptide FF-like peptides that attenuate morphine-induced antinociception

The two mammalian neuropeptides NPFF and NPAF have been shown to have important roles in nociception, anxiety, learning and memory, and cardiovascular reflex. Two receptors (FF1 and FF2) have been molecularly identified for NPFF and NPAF. We have now characterized a novel gene designated NPVF that encodes two neuropeptides highly similar to NPFF. NPVF mRNA was detected specifically in a region between the dorsomedial and ventromedial hypothalamic nuclei. NPVF-derived peptides displayed higher affinity for FF1 than NPFF-derived peptides, but showed poor agonist activity for FF2. Following intracerebral ventricular administration, a NPVF-derived peptide blocked morphine-induced analgesia more potently than NPFF in both acute and inflammatory models of pain. In situ hybridization analysis revealed distinct expression patterns of FF1 and FF2 in the rat central nervous system. FF1 was broadly distributed, with the highest levels found in specific regions of the limbic system and the brainstem where NPVF-producing neurons were shown to project. FF2, in contrast, was mostly expressed in the spinal cord and some regions of the thalamus. These results indicate that the endogenous ligands for FF1 and FF2 are NPVF- and NPFF-derived peptides, respectively, and suggest that the NPVF/FF1 system may be an important part of endogenous anti-opioid mechanism.     

Mice lacking the dopamine transporter display altered regulation of distal colonic motility

The mechanisms by which dopamine (DA) influences gastrointestinal (GI) tract motility are incompletely understood and complicated by tissue- and species-specific differences in dopaminergic function. To improve the understanding of DA action on GI motility, we used an organ tissue bath system to characterize motor function of distal colonic smooth muscle segments from wild-type and DA transporter knockout (DAT -/-) mice. In wild-type mice, combined blockade of D(1) and D(2) receptors resulted in significant increases in tone (62 +/- 9%), amplitude of spontaneous phasic contractions (167 +/- 24%), and electric field stimulation (EFS)-induced (40 +/- 8%) contractions, suggesting that endogenous DA is inhibitory to mouse distal colonic motility. The amplitudes of spontaneous phasic and EFS-induced contractions were lower in DAT -/- mice relative to wild-type mice. These differences were eliminated by combined D(1) and D(2) receptor blockade, indicating that the inhibitory effects of DA on distal colonic motility are potentiated in DAT -/- mice. Motility index was decreased but spontaneous phasic contraction frequency was enhanced in DAT -/- mice relative to wild-type mice. The fact that spontaneous phasic and EFS-induced contractile activity were altered by the lack of the DA transporter suggests an important role for endogenous DA in modulating motility of mouse distal colon.     

Mechanism of action of neurotensin at the ileocecal sphincter region

Neurotensin, a neuropeptide identified in the distal small intestine, plays an unclear role in ileocecal sphincter regional function. The purpose of this study was to determine the effect and mechanism of action of neurotensin on the feline ileocecal sphincter (ICS), proximal colon, and distal ileum. Intraluminal pressures were recorded at these sites in anesthetized cats after superior mesenteric artery injection of neurotensin. Dose dependent tonic and phasic contractions were seen at all sites. Peak pressure responses were seen at the maximal dose used and were greater for the ICS than the distal ileum and the proximal colon. The threshold dose for peak pressures for neurotensin was 0.05 microgram/kg for all sites with the maximal peak pressures occurring at the maximal dose used (100 micrograms/kg). The motility index (MI [number of contractions x mean amplitude of contractions]) was determined for three minutes before and after neurotensin injection. The change in the motility index after neurotensin increased at doses above 0.05 micrograms/kg for the ileum and the ICS and 0.25 microgram/kg for the colon. Maximal responses for the motility index were seen at 1 microgram/kg for the distal ileum, and 10 micrograms/kg for the ICS and the proximal colon, with the greatest response seen at the ICS. Neurotensin-induced ICS relaxation was seen at 1 microgram/kg (50 +/- 10%, p less than 0.01) in 33% of cats. The contractile responses of the distal ileum and the proximal colon were not inhibited by naloxone, trimethaphan, tetrodotoxin, or atropine. The ICS contractile response was decreased by tetrodotoxin by 53%, p less than 0.05. The alpha 2 antagonist, yohimbine reduced the neurotensin induced ICS contraction from 31.6 +/- 3.4 to 21.9 +/- 3.3 mm Hg, p less than 0.05. Prazosin had no effect on neurotensin-induced contractions. In the presence of cimetidine and diphenhydramine, trimethaphan did not affect the neurotensin-induced contractile response at all three sites. However, neurotensin inhibited contractions induced by trimethaphan alone at all three sites. Conclusions: 1. Neurotensin causes a dose-dependent contractile response at the distal ileum, ICS, and proximal colon. 2. Neurotensin has an inhibitory effect at all three sites. 3. The contractile response at the distal ileum and the proximal colon is mediated via smooth muscle receptors. 4. The contractile response of neurotensin at the ICS is mediated partly via alpha 2 receptors and partly via smooth muscle receptors.     

Neuropeptide FF and neuropeptide VF inhibit GABAergic neurotransmission in parvocellular neurons of the rat hypothalamic paraventricular nucleus

Neuropeptide FF (NPFF) and neuropeptide VF (NPVF) are octapeptides belonging to the RFamide family of peptides that have been implicated in a wide variety of physiological functions in the brain, including central autonomic and neuroendocrine regulation. The effects of these peptides are mediated via NPFF1 and NPFF2 receptors that are abundantly expressed in the rat brain, including the hypothalamic paraventricular nucleus (PVN), an autonomic nucleus critical for the secretion of neurohormones and the regulation of sympathetic outflow. In this study, we examined, using whole cell patch-clamp recordings in the brain slice, the effects of NPFF and NPVF on inhibitory GABAergic synaptic input to parvocellular PVN neurons. Under voltage-clamp conditions, NPFF and NPVF reversibly and in a concentration-dependent manner reduced the evoked bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) in parvocellular PVN neurons by 25 and 31%, respectively. RF9, a potent and selective NPFF receptor antagonist, blocked NPFF-induced reduction of IPSCs. Recordings of miniature IPSCs in these neurons following NPFF and NPVF applications showed a reduction in frequency but not amplitude, indicating a presynaptic locus of action for these peptides. Under current-clamp conditions, NPVF and NPFF caused depolarization (6-9 mV) of neurons that persisted in the presence of TTX but was abolished in the presence of bicuculline. Collectively, these data provide evidence for a disinhibitory role of NPFF and NPVF in the hypothalamic PVN via an attenuation of GABAergic inhibitory input to parvocellular neurons of this nucleus and explain the central autonomic effects of NPFF.     

Cholecystokinin stimulates neuronal receptors to produce contraction of the canine colon

The motor effects of cholecystokinin 26-33-amide (CCK octapeptide; CCK-OP) and several purported CCK receptor antagonists on canine colonic circular muscle were determined in pentobarbital anesthetized dogs. Intravenous injections of CCK-OP had no effect on colonic motility at doses that contracted the gallbladder, stomach and duodenum. CCK-OP delivered by intraarterial injection to a small segment of the proximal colon produced a dose related increase in colonic motility with one-half maximum response at 12 ng/Kg and maximum response at 50 ng/Kg. The effects of intraarterial injections of several established CCK-receptor antagonists on proximal colonic responses to intraarterial injections of CCK-OP were determined. Proglumide, 10 mg/Kg, did not produce colonic contractions itself, but antagonized CCK-OP-induced responses. Carbobenzyloxy (CBZ)-CCK27-32-amide antagonized CCK-OP-induced colonic responses and also had no effect on basal colonic motility (0.1-1 and 5 micrograms/Kg). Neither compound antagonized acetylcholine- induced colonic responses. Butoxycarbonyl (BOC)-CCK31-33-amide increased basal colonic motility, but did not alter CCK-OP-induced responses at doses of 0.1 and 0.2 mg/Kg. Dibutyryl-cGMP at a dose of 0.1 mg/Kg did not affect basal motility or CCK-OP-induced contractions. At a dose of 1.0 mg/kg it increased basal colonic motility but did not affect CCK-OP-induced contractions. Pentagastrin increased colonic motor activity only at a dose of 5 micrograms/Kg, i.a., a much higher dose than effective doses of CCK-OP. The mechanism of CCK-OP-induced colonic motor effects also was determined. Atropine sulfate, 100 micrograms/Kg, i.v. significantly reduced both intraarterial acetylcholine-and CCK-OP-induced maximum colonic contractions. Tetrodotoxin, at intravenous doses that completely block neuronal activity, did not affect maximum acetylcholine-induced contractions but practically eliminated maximum CCK-OP-induced maximum colonic responses. In conclusion, intraarterial CCK-OP produces circular muscle contraction of the canine proximal colon that is mediated by stimulation of specific CCK receptors which produce the release of acetylcholine from cholinergic enteric neurons. Proglumide and CBZ-CCK27-32-amide are effective CCK receptor antagonists at these colonic neuronal receptors.     

Contribution of NK(2) tachykinin receptors to propulsion in the rabbit distal colon

The role of the tachykinin neurokinin (NK)(2) receptors on rabbit distal colon propulsion was investigated by using two selective NK(2)-receptor antagonists, MEN-10627 and SR-48968. Experiments on colonic circular muscle strips showed that contractile responses to [beta-Ala(8)]NKA-(4-10) (1 nM-1 microM), a selective NK(2)-receptor agonist, were competitively antagonized by MEN-10627 (1-100 nM), whereas SR-48968 (0.1-10 nM) caused an insurmountable antagonism, thus confirming the difference in the mode of action of the two compounds. Colonic propulsion was elicited by distending a mobile rubber balloon with 0.3 ml (submaximal stimulus) or 1.0 ml (maximal stimulus) of water. The velocity of anal displacement of the balloon (mm/s) was considered the main propulsion parameter. At low concentrations (1.0-100 nM and 0.1-10 nM, respectively), MEN-10627 and SR-48968 facilitated the velocity of propulsion, whereas at high concentrations (100 nM and 1 microM, respectively) they decelerated propulsion. The excitatory and inhibitory effects of both antagonists were observed only with submaximal stimulus. We focused on the hypothesis that the facilitatory effect on propulsion may result from blockade of neuronal NK(2) receptors and the inhibitory effect from suppression of the excitatory transmission mediated by NK(2) receptors on smooth muscle cells. In the presence of N(G)-nitro-L-arginine (300 microM), a nitric oxide synthase inhibitor, MEN-10627, at a concentration (10 nM) that was found to accelerate propulsion in control experiments inhibited the velocity of propulsion. In the presence of threshold (1-10 nM) or full (1 microM) concentration of atropine, which inhibited to a great extent the velocity of propulsion, the inhibitory effect of MEN-10627 (1 microM) was markedly increased. In conclusion, in the rabbit distal colon NK(2) receptors may decelerate propulsion by activating a nitric oxide-dependent neuronal mechanism and may accelerate it by a postjunctional synergistic interaction with cholinergic muscarinic receptors.     

Central administration of neuropeptide FF and related peptides attenuate systemic morphine analgesia in mice

Neuropeptide FF (NPFF) belongs to an opioid-modulating peptide family. NPFF has been reported to play important roles in the control of pain and analgesia through interactions with the opioid system. However, very few studies examined the effect of supraspinal NPFF system on analgesia induced by opiates administered at the peripheral level. In the present study, intracerebroventricular (i.c.v.) injection of NPFF (1, 3 and 10 nmol) dose-dependently inhibited systemic morphine (0.12 mg, i.p.) analgesia in the mouse tail flick test. Similarly, i.c.v. administration of dNPA and NPVF, two agonists highly selective for NPFF(2) and NPFF(1) receptors, respectively, decreased analgesia induced by i.p. morphine in mice. Furthermore, these anti-opioid activities of NPFF and related peptides were blocked by pretreatment with the NPFF receptors selective antagonist RF9 (10 nmol, i.c.v.). These results demonstrate that activation of central NPFF(1) and NPFF(2) receptors has the similar anti-opioid actions on the antinociceptive effect of systemic morphine.     

Functional coordination of motor activity in colonic smooth muscles in rat experimental model

Spontaneous and electrically-elicited motor activity was recorded by triple organ bath in rat segment-model preparation as display of excitation of local nerve networks and ascending or descending reflex pathways underlying contractile potency and functional coordination of colonic longitudinal and circular muscles. Spontaneous high-amplitude contractions, but not relaxations, appeared synchronously in both muscles. Electrical field stimulation applied to proximal or distal part of segments elicited both tetrodotoxin (0.1 microM)-sensitive local motor responses of the stimulated part and ascending or descending motor responses of the contralateral, nonstimulated part of the preparations. Contractions characterized the local response of longitudinal muscle. The circular muscle responded with relaxation followed by contraction. Synchronous ascending contractions and descending contraction of the longitudinal muscle and relaxation followed by contraction of the circular muscle were observed when the middle part of segments was stimulated, thus indicating that locally-induced nerve excitation propagated via intrinsic ascending or descending nerve pathways that could be synchronously coactivated by one and the same stimulus. The ascending motor responses were more pronounced and the motor responses of longitudinal muscle were expressed more than those of circular muscle suggesting an essential role of ascending reflex pathways and longitudinal muscle in the coordinated motor activity of colon.     

Inhibition of peristaltic activity by cannabinoids in the isolated distal colon of mouse.

The effects of the cannabinoid receptor agonist Win 55,212-2 and of the competitive cannabinoid receptor antagonist SR 141716A on the electrically-evoked peristalsis of isolated distal colon of mouse were studied. Intraluminal pressure, longitudinal displacement, ejected fluid volume and changes in morphology of external intestinal wall were simultaneously recorded in the pre-drug period and in presence of Win 55,212-2 alone or in combination with SR 141716A. In the pre-drug period (control), peristaltic activity was characterised by regular, monophasic waves and the intraluminal content propelled towards anterograde (oro-aboral) direction with a propulsion velocity of 1.25 +/- 0.1 mm x s(-1). Pressure and shortening waves showed a peak amplitude of 2.44 +/- 0.32 kPa and 1.8 +/- 0.72 mm, respectively. The mean amount of fluid volume ejected during each contraction was 80 +/- 12.6 microl. The addition of Win 55,212-2 [10(-7)-10(-4) M] to the organ bath determined a dose-related attenuation of peristaltic activity consequent to the decrease of circular and longitudinal muscle strength. The decrease of contractile activity was followed by dose-dependent decrease of the amount of fluid ejected during peristalsis. The effects of Win 55,212-2 [10(-7)-10(-5) M] were prevented by SR 141716A, indicating the presence of cannabinoid CB1 receptors in the mouse distal colon. SR 141716A alone enhanced both tonic and phasic motor activities in the colonic longitudinal smooth muscle, suggesting that CB1 receptor antagonists could act either through antagonising the effect of endogenous CB1 receptor agonist or by an agonist effect on these receptors. The present results further support the hypothesis that cannabinoids perform a neuromodulatory role in various tracts of gastrointestinal system and first demonstrate their action also in the distal colon of rodents.     

Anti-opioid activities of NPFF1 receptors in a SH-SY5Y model

In order to elucidate the mechanisms of the neuronal anti-opioid activity of Neuropeptide FF, we have transfected the SH-SY5Y neuroblastoma cell line, which expresses mu- and delta-opioid receptors, with the human NPFF1 receptor. The SH1-C7 clone expresses high affinity NPFF1 receptors in the same range order of density as opioid receptors. Similarly to the opioids, acute stimulation with the NPFF1 agonist NPVF inhibits adenylyl cyclase activity and voltage-gated (N-type) Ca2+ currents and enhances the intracellular Ca2+ release triggered by muscarinic receptors activation. In contrast, preincubation of cells with NPVF decreases the response to opioids on both calcium signaling, thus reproducing the cellular anti-opioid activity described in neurons. SH1-C7 cells are therefore a suitable model to investigate the interactions between NPFF and opioid receptors.     

Adrenergic receptors mediating prostaglandin production in the rabbit vas deferens

Contractile and prostaglandin E (PGE)-producing effects of adrenergic agonists were compared in the rabbit isolated vas deferens to determine which adrenergic receptor(s) potentially could mediate neural responses. Additionally, interactions among receptors were elucidated by comparing responses to norepinephrine, phenylephrine and isoproterenol to those in the presence of selective adrenergic agonists or antagonists. Norepinephrine increased the force of muscle contraction and the immunoassayable PGE concentrations in a concentration-dependent manner with EC50's of 55 +/- 8 and 112 +/- 39 microM, respectively. Propranolol (10 microM) enhanced the contractile effects of norepinephrine (p less than 0.01) whereas yohimbine (100 microM) or prazosin (1 microM) reduced norepinephrine-induced contractions and PGE production (p less than 0.01). Propranolol did not alter the PGE production induced by norepinephrine. Metoprolol (100 microM) also enhanced contractile effects of norepinephrine (p less than 0.05). The beta adrenergic agonist, isoproterenol (100 nM), decreased the contractile, but not the PGE-producing, effects of phenylephrine (p less than 0.001). Isoproterenol, given alone, increased PGE concentrations and inhibited electrically-induced force generation in a concentration-dependent manner. These results are consistent with the presence of alpha receptors on the vas deferens which mediate smooth muscle contraction and PGE generation. Beta receptors which mediate relaxation and PGE production also are present. Tentative identification of the beta receptor subtype revealed the presence of a beta 1 receptor.     

Arecoline excites the colonic smooth muscle motility via M3 receptor in rabbits

Arecoline is an effective component of areca (betel nuts, a Chinese medicine named pinang or binglang). The purpose of this study was to investigate the effect of arecoline on the motility of distal colon in rabbits and its mechanisms involved. Strips of colonic smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were examined. The response of smooth muscle to arecoline in colonic strips was recorded. The effects of atropine, gallamine and 1,1-dimethyl-4-diphenylacetoxypiperidiniumiodide (4-DAMP) on arecoline-induced contraction were also observed. Arecoline (1 nM - 1 microM) produced a concentration-dependent contraction in both the longitudinal and the circular smooth muscle of rabbit colon. Atropine (10 microM) abolished the arecoline (80 nM)--induced contraction. M3 receptor antagonist, 4 - DAMP (0.4 microM), abolished the arecoline (80 nM)--related response, whereas M2 receptor antagonist, gallamine (0.4 microM), did not affect the effect of arecoline. These results suggest that arecoline excites the colonic motility via M3 receptor in rabbits.     

Pharmacological and biochemical characterization of bradykinin B2 receptors in the mouse colon: influence of the TNBS-induced colitis

This study analyzed bradykinin (BK)-evoked contractile responses in the mouse colon under normal and inflammatory conditions. BK and the preferential B(2) receptor agonists Hyp(3)-BK, Lys-BK, Met-Lys-BK and Tyr(8)-BK produced a marked and concentration-related contraction of the normal mouse colon, whereas the selective B(1) receptor agonist des-Arg(9)-BK had no effect. BK-induced contraction was concentration-dependently antagonized (in a non-competitive manner) by both B(2) receptor antagonists Hoe 140 and FR173657, but not the B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK. Analysis of the possible mechanisms implicated in the contractile responses of BK in the mouse colon revealed the involvement of the neural release of acetylcholine, the activation of L- and N-type voltage-gated calcium channels, and the release of neuropeptides, prostanoids and leukotrienes. The contraction induced by BK was markedly increased in preparations obtained from TNBS-treated mice. The up-regulation of B(2) receptors following the induction of colitis was confirmed with binding studies using [(3)H]-BK, which revealed a marked increase in B(2) receptor densities, without alterations of affinity. We provide convincing evidence on the relevance of B(2) receptors in the mouse colon under normal conditions, as well as under an inflammatory profile of colitis. Selective B(2) receptor antagonists might well represent rational therapeutic options for treating inflammatory bowel diseases.     

Calcitonin gene-related peptide (CGRP) inhibits contractions of the prostatic stroma of the rat but not the guinea-pig

This study investigated the presence and effects of calcitonin gene-related peptide (CGRP) within the rat and guinea-pig prostate glands. Immunohistochemical studies demonstrated that CGRP immunoreactive nerve fibres are sparsely distributed throughout the prostatic fibromuscular stroma in both species. These CGRP immunopositive nerve fibres shared a similar distribution profile but were not colocalized with tyrosine hydroxylase immunopositive nerve fibres which also innervate the prostatic stroma of these species. Nerve terminals within rat and guinea-pig prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). In guinea-pig preparations, application of human alpha-CGRP, rat adrenomedullin or rat amylin (0.1 nM-1 microM) had no effect on responses to field stimulation. In contrast, both rat and human alpha-CGRP (10 pM-300 nM), rat adrenomedullin (0.3 nM-1 microM) and rat amylin (3 nM-1 microM) concentration-dependently inhibited electrically evoked contractile responses in the rat prostate. The relative order of potency was rat alpha-CGRP=human alpha-CGRP>rat adrenomedullin>rat amylin. The inhibition by rat alpha-CGRP of field stimulation-induced contractions in the rat prostate was competitively antagonized by human CGRP((8-37)) (1, 3 and 10 microM) with a pA(2) of 6.20+/-0.13. Rat alpha-CGRP (10 nM) attenuated contractile responses of the rat prostate to exogenously added noradrenaline (1-100 microM). Inhibitory concentration-response curves to rat alpha-CGRP in rat prostates were unaffected by preincubation in either glibenclamide (10-100 microM), N-nitro-L-arginine methyl ester (L-NAME) (10 microM), bestatin (10 microM), captopril (10 microM) or phosphoramidon (3 microM). Our results indicate that CGRP-induced inhibition of electrically evoked contractions in the rat prostate occurs through activation of postjunctional CGRP(2) receptors which act independently of a K(ATP) channel or nitrergic mechanisms. Degradation of rat alpha-CGRP via peptidases does not appear to occur in the rat prostate.     

Short-chain fatty acids stimulate colonic transit via intraluminal 5-HT release in rats

We studied whether physiological concentration of short-chain fatty acids (SCFAs) affects colonic transit and colonic motility in conscious rats. Intraluminal administration of SCFAs (100-200 mM) into the proximal colon significantly accelerated colonic transit. The stimulatory effect of SCFAs on colonic transit was abolished by perivagal capsaicin treatment, atropine, hexamethonium, and vagotomy, but not by guanethidine. The stimulatory effect of SCFAs on colonic transit was also abolished by intraluminal pretreatment with lidocaine and a 5-hydroxytryptamine (HT)(3) receptor antagonist. Intraluminal administration of SCFAs provoked contractions at the proximal colon, which migrated to the mid- and distal colon. SCFAs caused a significant increase in the luminal concentration of 5-HT of the vascularly isolated and luminally perfused rat colon ex vivo. It is suggested that the release of 5-HT from enterochromaffin cells in response to SCFAs stimulates 5-HT(3) receptors located on the vagal sensory fibers. The sensory information is transferred to the vagal efferent and stimulates the release of acetylcholine from the colonic myenteric plexus, resulting in muscle contraction.     

Effects of econazole on receptor-operated and depolarization-induced contractions in rat isolated aorta

In our previous study, econazole caused a decrease in serum nitrite levels in septic mice in vivo, but it enhanced the mortality rate. The aim of the study was to investigate the in vitro effects of econazole on receptor-operated and depolarization-induced contractions on endothelium-intact and -denuded rat isolated aorta. Econazole (0.1, 1 and 10 microM) significantly inhibited receptor-operated (phenylephrine, Phe) and depolarization (KCl)-induced contractions of endothelium-intact or -denuded rings in a noncompetitive and concentration-dependent manner. Removal of endothelium changed the pD'2 values only for KCl-induced responses. The pD'2 values of L-type calcium channel blocker nifedipine were significantly higher than the econazole on Phe concentration-response curves in endothelium-intact and -denuded rings. Econazole caused a biphasic response in precontracted by Phe or KCl in endothelium-intact and -denuded rings, first a transient contraction following sustained relaxation. Removal of endothelium did not affect the contractile responses induced by Phe. The contractile responses induced by 10 microM econazole in the KCl-precontracted rings were antagonized by the treatment of alpha-adrenergic receptor antagonist, phentolamine (10 microM). Deendothelization was significantly increased the IC50 values of econazole obtained from Phe- and KCl-precontractions. The relaxations induced by 10 microM econazole in endothelium-intact rings precontracted with Phe or KCl were not changed by NO synthase inhibitor, L-N(G)-nitroarginine (100 microM). The IC50 values of econazole were significantly higher than nifedipine in endothelium-intact and -denuded rings. These results suggest that econazole is a noncompetitive antagonist on alpha1-adrenoceptor-mediated and depolarization-induced contractions in rat isolated aorta by inhibiting Ca2+ entry through L-type calcium channels, and the endothelium seems to modulate vascular responses induced by this agent. The vascular effects of econazole may limit the usage of this agent in septic shock.     

Effect of lignocaine on intestinal smooth muscle of rat ileum

The effect of lignocaine on tone and contractility of intestinal smooth muscle, and on contractures produced by ACh or TEA, was studied in isolated ileum of the rat. Lignocaine (0.1-100 microM) produced concentration-dependent contractures in the rat ileum. In low concentrations, lignocaine increased the amplitude of spontaneous contractions and contractions produced by transmural stimulation. High concentrations of lignocaine abolished all contractile responses and produced a marked contracture in rat ileum. Lignocaine (10 microM) also reduced the contractures produced by ACh (0.01-10 microM). In contrast, the contractures produced by TEA (0.1-10 mM) were markedly increased by lignocaine. Furthermore, the contracture produced by lignocaine was reduced by lowering the external calcium from 2.5 mM to 1.5 mM. It was concluded that lignocaine in moderate and high concentrations produces a contracture in rat intestinal smooth muscle. Whereas lignocaine reduces the ACh-induced contracture, it increases that produced by TEA in the same preparation. The results further suggest that lignocaine modifies cholinergic responses and affects excitation-contraction coupling in rat intestinal smooth muscle.     

Role of insulin preincubation in the contractile reactivity of rat aortic rings.

Preincubation with physiological concentrations of insulin affects contractile reactivity of isolated smooth muscle cells. We studied the effects of insulin on intact aortic rings of Wistar rats preincubated 1-2 h with 240 pM (I1) and 960 pM (I2) insulin with and without NO synthesis inhibition by N(omega)-nitro-L-arginine methyl ester (L-NAME). Resting force was tripled by 0.1 mM L-NAME in control (C) and I1 groups, but not in I2 groups. I1 treatment decreased the tachyphylaxis to two successive 1 microM arginine vasopressin (AVP) stimulations. Single contractions elicited by 1 microM AVP, 1 microM angiotensin II (AngII), or 0.01 microM endothelin (ET1) were not affected by insulin preincubation in either maximal force (Fmax) or relaxation times. L-NAME enhanced Fmax of AngII contractions by about 75% in C, 120% in I1, and 74% in I2 groups; accordingly, it augmented the final steady-state force in C and I1 but not in I2. Similarly, L-NAME increased Fmax (30-40%) of AVP and ET1 contractions in C and I1 groups but failed to do so in contractions of I2 group. Results obtained with 10 microM indomethacin suggest that this is due to insulin stimulation of prostacyclin effects.