A synthetic VIP peptide analogue inhibits neutrophil recruitment in rat airways in vivo

Currently, there is no effective pharmacotherapy against exaggerated mobilisation of neutrophils in human airway diseases such as chronic obstructive pulmonary disease and asthma. We evaluated the effect of two synthetic vasoactive intestinal peptide (VIP)-like analogues on cytokine-induced neutrophil recruitment in airways in vivo. Recombinant interleukin (IL)-1 beta was administered intratracheally (i.t.) to intubated, spontaneously breathing Sprague-Dawley rats. The rats were pretreated either with a VIP synthetic peptide analogue, a pituitary adenylate cyclase-activating peptide (PACAP)-1-27 synthetic analogue, the beta(2)-adrenoceptor agonist salbutamol or vehicle, systemically or locally. Differential cell counts were performed on bronchoalveolar lavage fluid (BALf) cytospins. Effects on mean arterial blood pressure (MAP) were monitored in separate experiments. Systemic administration of the VIP analogue, the PACAP analogue and salbutamol attenuated the cytokine-induced increase in BALf neutrophil number. Local administration of the VIP analogue and salbutamol, but not the PACAP analogue, also decreased the neutrophil number in BALf. Local administration of the VIP analogue and salbutamol caused a transient decrease in MAP. Systemic or local administration of a synthetic VIP peptide analogue inhibits cytokine-induced neutrophil recruitment in airways in vivo. This action is exerted without severe, sustained cardiovascular side effects, and deserves to be further evaluated in obstructive pulmonary diseases in human.     

Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus

The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N(G)-nitro arginine (L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by alpha-chymotrypsin. alpha-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by alpha-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe6,Leu17]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO.     

Functional effects of 20-HETE on human bronchi: hyperpolarization and relaxation due to BKCa channel activation

Airway smooth muscle (ASM) metabolizes arachidonic acid (AA) through various enzymatic pathways, including cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to delineate the mode of action of 20-HETE in human ASM cells. Isometric tension measurements demonstrated that 20-HETE induced a concentration-dependent relaxant effect in ASM on bronchi precontracted with either methacholine or AA. Relaxing effects of 20-HETE on resting tone were prevented by 10 nM iberiotoxin (IbTx), a BK(Ca) channel inhibitor. Microelectrode measurements showed that exogenous additions of 20-HETE (0.1-10 microM) hyperpolarized the membrane potential of human ASM cells. This concentration-dependent electrophysiological effect induced by the eicosanoid was prevented by 10 nM IbTx. Complementary experiments, using the planar lipid bilayer reconstitution technique, demonstrated that 20-HETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentrations. Together, these results indicate that 20-HETE-dependent activation of BK(Ca) channels is responsible for the hyperpolarization and controlled relaxation of ASM in human distal bronchi.     

Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery

Although airway and pulmonary vessel tone are regulated predominantly by cholinergic and adrenergic impulses, biologically active peptides such as calcitonin gene-related peptide (CGRP) may significantly influence human smooth muscle tone in normal and pathophysiological states. In the present study, the expression of CGRP and its receptor CGRPR-1 and the biological effect of the peptide were investigated in human airways and pulmonary arteries. Immunohistochemistry revealed the presence of CGRP in human airway nerves and neuro-epithelial cells, whereas the receptor was found in epithelial cells and smooth muscle myocytes of the bronchi and in pulmonary artery endothelium. On precontracted bronchi (3-4 mm in diameter) alpha-CGRP (0.01-10 nM) caused a concentration-dependent contraction on epithelium-denuded bronchi, whereas no significant effect was recorded in bronchi with intact epithelium. In pulmonary arteries (2-6 mm in diameter), alpha-CGRP caused a concentration-dependent relaxation of endothelium intact and denuded vessels. Pre-treatment with indomethacin, but not with l-NAME, prevented the relaxation induced by alpha-CGRP in pulmonary arteries suggesting that prostaglandins but not nitric oxide (NO) are involved in the intracellular signal transduction pathway. The effects induced by alpha-CGRP in bronchi and vessels were prevented by application of the antagonist CGRP((8-37)). In summary, the present studies examined the biological function of CGRP in human airways and demonstrated a constrictory effect of CGRP only in epithelium-denuded airway smooth muscle indicating an alteration of CGRP airway effects in respiratory tract pathological states with damaged epithelium such as chronic obstructive pulmonary disease or bronchial asthma.     

Pituitary adenylate cyclase activating peptide (PACAP) in guinea-pig lung: distribution and dilatory effects.

The lower airways of guinea-pigs were analyzed for pituitary adenylate cyclase activating peptide (PACAP) using immunocytochemistry. In the trachea a moderate supply of PACAP-immunoreactive nerve fibers occurred around smooth muscle bundles, glands and small blood vessels. In the lung, PACAP-immunoreactive nerve fibers were distributed around small glands and bronchi. A rich supply of PACAP immunoreactive nerve fibers was found around blood vessels in the lungs. PACAP-suppressed smooth muscle responses were analysed using isolated circular segments of trachea, pulmonary arteries and aorta of guinea-pigs. In both airways and arteries PACAP caused a concentration-dependent relaxation of precontracted segments. The maximal relaxation effects were more pronounced in the airways than in the arteries while the order of potency was aorta greater than pulmonary artery greater than trachea. The effect of PACAP was compared to those of acetylcholine (ACh) and vasoactive intestinal peptide (VIP). In the pulmonary artery the vasomotor responses expressed as maximal dilatation had the order: ACh greater than VIP = PACAP while the order of potency was PACAP = VIP greater than ACh. In the trachea, PACAP was slightly more potent than VIP. The relaxatory responses to PACAP in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine, cimetidine and Spantide. Removal of the endothelium abolished PACAP-induced vascular relaxation. Conceivably, PACAP-containing nerve fibers play a role in the regulation of airway resistance and local blood flow.     

Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9]VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, "hop" splice variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and alpha-helical regions of PACAP-27 are required for initiating gallbladder contraction.     

Effect of lithium on endothelium-dependent and neurogenic relaxation of rat corpus cavernosum: role of nitric oxide pathway.

INTRODUCTION: Some studies have reported erectile dysfunction in patients receiving lithium through a mechanism that has not yet been defined. The aim of the present study was to verify the effect of acute lithium administration on the nonadrenergic noncholinergic (NANC)- and endothelium-mediated relaxation of rat isolated corpus cavernosum. MATERIALS AND METHODS: The isolated rat corporeal strips were precontracted with phenylephrine hydrochloride (7.5 microM) and electrical field stimulation (EFS) was applied at different frequencies (2, 5, 10, and 15 Hz) to obtain NANC-mediated relaxation or relaxed by adding cumulative doses of acetylcholine (10nM-1mM) to obtain endothelium-dependent relaxation in the presence or absence of lithium (0.3, 0.5, 1, and 5mM). Also, effects of combining lithium (0.3mM) with 30 nM and 0.1 nM L-NAME (an NO synthase inhibitor) on NANC- and acetylcholine-mediated relaxation was investigated, respectively. Moreover, effects of combining lithium (1mM) with 0.1mM and 10 microM L-arginine (a precursor of NO) on NANC- and endothelium-mediated relaxation was assessed, respectively. Also, the effect of lithium (1mM) on relaxation to sodium nitroprusside (SNP; 1nM-1mM), an NO donor, was investigated. RESULTS: The NANC-mediated relaxation was significantly (P<0.001) reduced by 1 and 5mM, but not by 0.3 and 0.5mM lithium. Lithium significantly (P<0.001) attenuated the maximum response to acetylcholine in a concentration-dependent manner. Combination of lithium (0.3mM) with 30 and 0.1 nM L-NAME, which separately had a minimum effect on NANC- and endothelium-mediated relaxation, significantly (P<0.001) reduced the NANC- and endothelium-mediated relaxation, respectively. Although L-arginine at 10 microM and 0.1mM did not alter the relaxant responses to acetylcholine and EFS, it improved the inhibition by lithium (1mM) of relaxant responses to acetylcholine and EFS, respectively. Also, SNP produced similar concentration-dependent relaxations from both groups. DISCUSSION: Our experiments indicated that lithium likely by interfering with NO pathway in both endothelium and nitrergic nerve can result in impairment of both the endothelium- and NANC-mediated relaxation of rat corpus cavernosum.     

Contribution of glibenclamide-sensitive, ATP-dependent K+ channel activation to acetophenone analogues-mediated in vitro pulmonary artery relaxation of rat

Compared to the currently available therapeutic drugs for peripheral vascular diseases, agents that are selective for relaxing pulmonary circulation are scarce. The present study was undertaken, using isometric tension change measurement and whole-cell patch-clamp electrophysiology methods, to evaluate the vascular relaxation effect and the underlying mechanisms involved of two naturally found alkaloids: paeonol (2-hydroxy-4-methoxy-acetophenone), acetovanillone (4-hydroxy-3-methoxy-acetophenone) and the non-substituted analogue acetophenone on pulmonary artery of Sprague-Dawley rats. Cumulative administration (3 microM-1 mM) of acetophenone analogues resulted in a concentration-dependent relaxation of phenylephrine (1 microM) pre-contracted pulmonary artery. A relative order of inhibitory potency, estimated by comparing the concentration at which a 50% relaxation of phenylephrine-induced contraction observed was: acetovanillone > paeonol > acetophenone. Endothelial denudation and inhibition of nitric oxide synthase (with 20 microM N(G)-nitro-L-arginine methyl-ester) only moderately suppressed (17.6 +/- 4.2%) acetovanillone- but not paeonol- or acetophenone-mediated maximum relaxation. Glibenclamide (3 microM, an ATP-sensitive K(+) (IK(ATP)) channel blocker) markedly attenuated all acetophenone analogues-mediated endothelium-independent relaxation. Neither cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL 12330A, 10 microM), iberiotoxin (300 nM), 4-aminopyridine (3 mM), (+/-)-propranolol (1 microM, a non-selective beta-adrenoceptor blocker) nor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (3 microM, a guanylate cyclase inhibitor) altered endothelium-independent relaxation. In electrophysiological experiments using single pulmonary artery smooth muscle cells, acetovanillone, paeonol, acetophenone and cromakalim activated glibenclamide-sensitive, IK(ATP) channels. In conclusion, our results demonstrate that acetophenone analogues caused pulmonary artery relaxation through opening of IK(ATP) channels. In addition, acetovanillone-mediated pulmonary artery relaxation is partly depended on nitric oxide released from endothelium.     

Effects of cod bradykinin and its analogs on vascular and intestinal smooth muscle of the Atlantic cod, Gadus morhua.

The effects of [Arg(0),Trp(5),Leu(8)]-BK (cod [Arg(0)]BK) on vascular preparations from branches of the cod celiac artery and on longitudinal smooth muscle preparations from the cod intestine were investigated. Cod [Arg(0)]BK (3 x 10(-8) M) caused a relaxation of the celiac artery precontracted with adrenaline. The relaxation was abolished by the cyclooxygenase inhibitor indomethacin, suggesting that the effect is mediated through the release of prostaglandins, but there was no evidence for the involvement of leukotrienes or nitric oxide in the response. In the intestinal preparations, cod [Arg(0)]BK produced concentration-dependent contractions (pD(2) = 8.28 +/- 0.16). Experiments with N-terminally and C-terminally truncated analogs and with alanine-substituted analogs of cod [Arg(0)]BK demonstrate that the central amino acid Gly(4) and the C-terminal amino acids Leu(8) and Arg(9) are the most important in determining the conformation of the peptide that interacts with the receptor. The results indicate that the ligand binding properties of the cod BK receptor are considerably different from the receptor present in trout tissues and may resemble those of the mammalian B(2) receptor more closely.     

Structure-activity relationship of synthetic truncated analogues of vasoactive intestinal peptide (VIP): an enhancement in the activity by a substitution with arginine

In order to develop potent shortened analogues of vasoactive intestinal peptide (VIP), the structure-activity relationship of C-terminally truncated analogues of VIP was investigated by examining the binding activity to rat lung VIP receptors and relaxation of smooth muscle in isolated mouse stomach. VIP(1-27) showed VIP receptor binding activity comparable to that of VIP but the activity of VIP(1-26) was reduced to one-third of VIP. The receptor binding activity of VIP(1-26) to VIP(1-23) was reduced in proportion to the decrease in amino acid residues. There was a significant correlation between the number of amino acid residues and VIP receptor binding activities of VIP and its C-terminally truncated analogues. VIP(1-22) and VIP(1-21) exhibited little binding activity even at high concentrations, suggesting the requisite of 23 amino acid residues as the minimal essential sequence for the conservation of VIP receptor binding activity. The chemical modification of VIP(1-23) generated a potent analogue, [Arg(15, 20, 21), Leu(17)]-VIP(1-23), that displayed a 22-fold higher receptor binding activity and 1.6-fold more potent relaxation of mouse stomach than VIP(1-23) did. In conclusion, it was shown that [Arg(15, 20, 21), Leu(17)]-VIP(1-23) could be a relatively potent and stable agonist of VIP receptors. The present study has provided further insight into the structure-activity relationship of VIP to generate novel shortened VIP analogues having a high affinity to VIP receptors and potent pharmacological activity.     

Long-acting analogue of vasoactive intestinal peptide, [R15, 20, 21, L17]-VIP-GRR (IK312532), protects rat alveolar L2 cells from the cytotoxicity of cigarette smoke

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in numerous biological responses. We previously reported that the replacement of Lys by Arg, and Met by Leu in VIP (IK312532; [Arg15, 20, 21, Leu17]-VIP) resulted in a significant improvement in metabolic stability and biological activity. In the present study, we investigated the effect of VIP and its related peptides including long-acting VIP derivative (IK312532) and PACAP27 on the cytotoxicity of cigarette smoke extract (CSE), a causative factor of chronic obstructive pulmonary disease (COPD), in rat alveolar L2 cells. RT-PCR displayed the dominant expression of mRNA for the VIP-specific VPAC2 receptor in L2 cells, and VIP and the related peptides showed the specific binding activity and potent stimulation of adenylate cyclase. CSE at a concentration of 0.1% or higher induced significant apoptotic death of L2 cells. Interestingly, the addition of neuropeptides at a concentration of 10(-11) M or higher in L2 cells with CSE (0.25%) resulted in significant attenuation of cell death with the deactivation of CSE-evoked caspase-3 activity. IK312532 was much stable against the enzymatic digestion compared to VIP, and the protective effect of IK312532 was 1.6-fold higher than that of VIP. Taken together with our previous report showing that IK312532 has long-acting relaxant activity in the lung, IK312532 may be a potential candidate for drug treatment of asthma and COPD.     

Changes in mechanism of PACAP-induced relaxation in longitudinal muscle of the distal colon of Wistar rats with age

Mechanisms of relaxation of longitudinal muscle of the distal colon induced by exogenously added pituitary adenylate cyclase activating peptide (PACAP) were studied in 2- to 30-week-old Wistar rats. Exogenous PACAP induced very significant relaxation of the longitudinal muscle in 2-week-old rats, but this effect decreased significantly with age. The cyclic AMP-cyclic AMP-dependent protein kinase (PKA) pathway and the tyrosine kinase-small conductance Ca2+-activated K+ channel (SK channel) pathway were found to be involved in the mechanism of PACAP-induced relaxation. In 2-week-old rats, PACAP-induced relaxation was significantly inhibited by tetrodotoxin (TTX). Since relaxation was also significantly inhibited by NG-nitro-L-arginine (N5-nitro-amidino-L-2,5-diamino-pentanoic acid: L-NOARG), the neurogenic effect of PACAP seems to be mediated mainly through nitric oxide neurons. In 8-week-old rats, L-NOARG and TTX had little effect on PACAP-induced relaxation, suggesting that the relaxant effect in 8-week-old rats is a direct action on longitudinal smooth muscle cells. Changes in the mechanisms of PACAP-induced relaxation with age were examined in the distal colon in relation to changes in the neurogenic and the direct effects of PACAP. The neurogenic effect in the exogenous PACAP-induced relaxation of the longitudinal muscle of the Wistar rat distal colon is dominant in tissue isolated from 2-week-old and lost in tissue isolated from 8-week-old rats.     

Relaxing effects of 5-oxo-ETE on human bronchi involve BK Ca channel activation

The present study investigated the ability of 5-oxo-EicosaTetraEnoic acid (5-oxo-ETE) for modulating airway smooth muscle (ASM) tone in human bronchi. 5-Oxo-ETE induced a concentration-dependent relaxing effect on human bronchi pre-contracted with methacholine (MCh) and arachidonic acid (AA). This relaxing response was highly sensitive to Iberiotoxin (IbTx), a large conducting Ca(2+)-activated K(+) channel (BK(Ca)) inhibitor. Furthermore, microelectrode measurements revealed that 5-oxo-ETE (0.1-10 microM) hyperpolarizes the membrane potential of human bronchial ASM cells. These hyperpolarizing effects were also inhibited in the presence of 10nM IbTx. Lastly, 5-oxo-ETE was shown to directly activate reconstituted BK(Ca) channels derived from human airway smooth muscles. In summary, the 5-oxo-ETE eicosanoid activates a specific K(+) conductance, involved in membrane hyperpolarization, which in turn reduces Ca(2+) entry and facilitates relaxation of smooth muscle cells.     

Nitric oxide involvement in the effect of acute lithium administration on the nonadrenergic noncholinergic-mediated relaxation of rat gastric fundus

INTRODUCTION: Lithium has largely met its initial promise as the first drug to be discovered in the modern era of psychopharmacology. However, the mechanism for its action remains an enigma. The aim of the present study was to verify the effect of acute lithium administration on the nonadrenergic noncholinergic (NANC)-mediated relaxation of rat isolated gastric fundus and to evaluate the role of nitric oxide pathway in this manner. MATERIALS AND METHODS: The isolated rat gastric fundus strips were precontracted with 0.5 microM serotonin and electrical field stimulation (EFS) was applied at 5 Hz frequency to obtain NANC-mediated relaxation in the presence or absence of lithium (0.1, 0.5, 1 and 5 mM). Also, effects of combining lithium (0.1 mM) with the NO synthase (NOS) inhibitor L-NAME (0.03 microM) or the guanylyl cyclase inhibitor ODQ (1 microM) on relaxant responses to EFS was investigated. Moreover, effects of combining lithium (1 mM) with 0.1 mM L-arginine (a precursor of NO) on neurogenic relaxation were assessed. Also, the effect of lithium (1 mM) on relaxation to sodium nitroprusside (SNP; 1 nM-0.1 mM) and glyceryltrinitrate (GTN; 0.1-10 microM) was investigated. RESULTS: The NANC-mediated relaxation was significantly (P<0.001) reduced by lithium in a dose- and time-dependent manner. Combination of lithium (0.1 mM) with L-NAME (0.03 microM), which separately had partial inhibitory effect on relaxations, significantly (P<0.001) reduced the NANC-mediated relaxation of gastric fundus. ODQ (1 microM) significantly inhibited the neurogenic relaxations in the presence or absence of lithium (0.1 and 1 mM). Although L-arginine at 0.1 mM had no effect on relaxation to EFS, it prevented the inhibition by lithium (1 mM) of relaxant responses to EFS. Also, SNP and GTN produced concentration-dependent relaxation in precontracted rat gastric fundus which was not altered by lithium incubation (1 mM). DISCUSSION: Our experiments indicated that lithium likely by interfering with L-arginine/NO pathway in nitrergic nerve can result in impairment of NANC-mediated relaxation of rat gastric fundus.     

Mechanisms underlying the relaxation induced by isokaempferide from Amburana cearensis in the guinea-pig isolated trachea

The present study examines possible mechanisms by which the flavonoid isokaempferide (IKPF; 5,7,4'-trihydroxy-3-methoxyflavone) from Amburana cearensis, a Brazilian medicinal plant popularly used as bronchodilator, induces relaxation of guinea-pig isolated trachea. In the trachea (with intact epithelium) contracted by carbachol, IKPF (1-1000 microM) caused a graded relaxation, and the epithelium removal increased the sensitivity of the airway smooth muscle to IKPF (EC50, in intact tissue: 77.4 [54.8-109.2] microM; in denuded epithelium: 15.0 [11.3-20.1] microM). The IKPF-induced relaxation was inhibited in 41% by the nitric oxide (NO) synthase inhibitor L-NAME (100 microM); in 31% and 50% by the soluble guanylate cyclase (sGC) inhibitor ODQ (3 and 33 microM); by propranolol (31%) and also by capsaicin (37%). In the trachea pre-contracted by 40 mM KCl the pre-incubation with glibenclamide (33 microM) or iberiotoxin (IbTX, 0.1 microM), selective K(+) channel inhibitors, inhibited the IKPF-induced relaxation by 39% and 38%, respectively. On the other hand, 4-aminopyridine (100 microM), a nonselective K(+) channel antagonist, did not significantly influence the effect of IKPF, while IbTX induced a rightward displacement of the IKPF concentration-response curve. However, in muscle pre-contracted with 120 mM KCl the relaxant effect of IKPF was significantly reduced and not affected by glibenclamide. In conclusion, these results indicate a direct and epithelium-independent relaxant effect of IKPF on smooth muscle fibers. Although this IKPF relaxant action seems to be multi-mediated, it occurs via both Ca(2+) and ATP-sensitive K(+) channels, but some other possible mechanisms unrelated to K(+) channels cannot be excluded.     

Role for pituitary adenylate cyclase activating polypeptide in cystitis-induced plasticity of micturition reflexes

Pituitary adenylate cyclase activating polypeptide (PACAP) peptides are expressed and regulated in sensory afferents of the micturition pathway. Although these studies have implicated PACAP in bladder control, the physiological significance of these observations has not been firmly established. To clarify these issues, the roles of PACAP and PACAP signaling in micturition and cystitis were examined in receptor characterization and physiological assays. PACAP receptors were identified in various tissues of the micturition pathway, including bladder detrusor smooth muscle and urothelium. Bladder smooth muscle expressed heterogeneously PAC(1)null, PAC(1)HOP1, and VPAC(2) receptors; the urothelium was more restricted in expressing preferentially the PAC(1) receptor subtype only. Immunocytochemical studies for PAC(1) receptors were consistent with these tissue distributions. Furthermore, the addition of 50-100 nM PACAP27 or PACAP38 to isolated bladder strips elicited transient contractions and sustained increases in the amplitude of spontaneous phasic contractions. Treatment of the bladder strips with tetrodotoxin (1 muM) did not alter the spontaneous phasic contractions suggesting direct PACAP effects on bladder smooth muscle. PACAP also increased the amplitude of nerve-evoked contractions. By contrast, vasoactive intestinal polypeptide had no direct effects on bladder smooth muscle. In a rat cyclophosphamide (CYP)-induced cystitis paradigm, intrathecal or intravesical administration of PAC(1) receptor antagonist, PACAP6-38, reduced cystitis-induced bladder overactivity. In summary, these studies support roles for PACAP in micturition and suggest that inflammation-induced plasticity in PACAP expression in peripheral and central micturition pathways contribute to bladder dysfunction with cystitis.     

Ursolic acid mediates the vasorelaxant activity of Lepechinia caulescens via NO release in isolated rat thoracic aorta

We have determined that the methanolic extract of L. caulescens (MELc) produced a significant vasodilator effect in a concentration-dependent and endothelium-dependent manner. This relaxation was blocked by N(omega)-nitro-L-arginine methylester (L-NAME), indicating that MELc vasodilator properties are endothelium mediated due to liberation of nitric oxide (NO). In this paper we aimed to corroborate its mode of action. MELc effects on noradrenaline (NA)-induced contraction in isolated rat aortic thoracic rings with endothelium (+E), in the presence of atropine (0.1 microM) and 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 1 microM) were conducted. MELc relaxation curve was significantly shifted to the right in the presence of ODQ and atropine, thus confirming that its mode of action is related with activation of nitric oxide synthase (NOS) and the consequent increment in NO formation. Bio-guided study of MELc allowed the isolation of ursolic acid (UA, 50 mg) and ursolic-oleanolic acids mixture [UA/OA (7:3), 450 mg]. The relaxant effect of UA (0.038-110 microM) was evaluated in functional experiments. UA induced a significant relaxation in a concentration- and endothelium-dependent manner (IC(50)=44.15 microM) and did not produce a vasorelaxant effect on contraction evoked by KCl (80 mM). In addition, NA-induced contraction was significantly displaced to the right by UA (30 microM). In order to determine its mode of action, UA-induced relaxant effect was evaluated in the presence of atropine (0.1 microM), indomethacin (10 microM), L-NAME (100 microM) and ODQ (1 microM). Relaxation was blocked by L-NAME and ODQ. On the other hand, UA (3 microM) provoked a significant displacement to the left in the relaxation curve induced by sodium nitroprusside (SNP, 0.32 nM to 0.1 microM), but it was not significant in the presence of Carbamoyl choline (carbachol, 1 nM to 10 microM). These results indicate that UA-mediated relaxation is endothelium dependent, probably due to NO release, and the consequent activation of vascular smooth muscle soluble guanylate cyclase (sGC), a signal transduction enzyme that forms the second messenger cGMP.     

Evidence of relaxant effect of omeprazole in rabbit corpus cavernosum in vitro

The present experiments were designed to investigate the effects of omeprazole, a H(+)-K+ ATPase inhibitor, on corporal smooth muscle tone in vitro. All spontaneous contractile activity in the corpus cavernosum was blocked following omeprazole (0.1 mM-1 mM) administration. However atropine (1 microM), Nw-nitro L-arginine methyl ester (L-NAME, 30 microM) or indomethacin (10 microM) did not affect the spontaneous contraction. Omeprazole (10 microM-1 mM) concentration-dependently induced relaxation in corporal smooth muscle precontracted with 10 microM phenylephrine or 80 mM KCl. Pretreatment of corporal tissue with L-NAME (30 microM), indomethacin (10 microM), ammonium chloride (7.5 mM), sodium acetate (7.5 mM), tetraethyl ammonium chloride (0.5 mM) or glibenclamide (1 microM) had no effect on the omeprazole induced relaxant responses. Nimodipine, an L-type Ca++ channel blocker, relaxed corporal strips precontracted with 80 mM KCl. Collectively, these results indicate that the inhibition of spontaneous contraction and the relaxation of precontracted corporal smooth muscle by omeprazole is probably mediated by the blockade of calcium channels. Further work is needed to determine the cellular mechanism(s) of action by which omeprazole acts on corpus cavernosum smooth muscle.     

Time-dependent alteration in cromakalim-induced relaxation of corpus cavernosum from streptozocin-induced diabetic rats

The purpose of the present study was to investigate the relaxant responses to the ATP-sensitive potassium (K(ATP)) channel opener cromakalim in corpus cavernosum strips from 1-, 2-, 4-, 6-, and 8-week streptozocin-induced diabetic rats. Cromakalim (1 nM-0.1 mM) produced concentration-dependent relaxation in phenylephrine (7.5 microM)-precontracted isolated rat corporal strips. Compared with age-matched control animals, a significant enhancement in cromakalim-induced relaxation of corpus cavernosum was observed in 2-week diabetic animals, whereas the relaxant responses to cromakalim were decreased in 6-and 8-week diabetic animals. However, the cromakalim-induced relaxation was not altered in either 1-week or 4-week rat corporal strips in comparison with corresponding age-matched non-diabetic groups. Preincubation with the K(ATP) channel blocker glibenclamide (10 microM) significantly inhibited the cromakalim-induced relaxation in both non-diabetic and diabetic rat corpus cavernosum, but neither the voltage-dependent K(+) channel (K(V)) antagonist 4-aminopyridine (1 mM) nor the calcium-activated K(+) channel (K(Ca)) antagonist charybdotoxin (0.1 microM) had significant effect on cromakalim-induced relaxation in both control and diabetic rat corporal strips. Relaxation responses to the nitric oxide donor sodium nitroprusside (1 nM-0.1 mM) in diabetic rat corpus cavernosum were similar to that of age-matched controls. These data demonstrated that the relaxant responses to cromakalim were altered in diabetic cavernosal strips in a time dependent manner, suggesting that the period of diabetes mellitus may play a key role in the K(ATP) channels function in rat corpus cavernosum.     

PACAP-27 tyrosine phosphorylates mitogen activated protein kinase and increases VEGF mRNAs in human lung cancer cells

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on human lung cancer cell line NCI-1299 mitogen activated protein kinase (MAPK) tyrosine phosphorylation and vascular endothelial cell growth factor (VEGF) expression were investigated. PACAP-27 (100 nM) increased MAPK tyrosine phosphorylation 3-fold, 5 min after addition to NCI-H1299 cells. PACAP caused tyrosine phosphorylation in a concentration-dependent manner being half-maximal at 10 nM PACAP-27. PACAP-27 or PACAP-38 (100 nM) but not PACAP28-38 or VIP caused increased MAPK tyrosine phosphorylation using NCI-H1299 cells. Also, the increase in MAPK tyrosine phosphorylation caused by PACAP-27 was totally inhibited by 10 microM PACAP(6-38), a PAC(1) receptor antagonist or 10 microM PD98059, a MAPKK inhibitor. These results suggest that PAC(1) receptors regulate tyrosine phosphorylation of MAPK in a MAPKK-dependent manner. PACAP-27 (100 nM) caused increased VEGF mRNA in NCI-H1299 cells after 8 h. The increase in VEGF mRNA caused by PACAP-27 was partially inhibited by PACAP(6-38), PD98059 and H-89. Addition of VIP to NCI-H1299 cells caused increased VEGF mRNA, which was totally inhibited by H89, a PKA inhibitor. These results suggest that PAC(1) and VPAC(1) receptors regulate VEGF expression in lung cancer cells.