Intracellular Ca2+ elevation and contraction due to prostaglandin F2alpha in rat aorta

Prostaglandin F2alpha was tested to determine (a) whether its effect on intracellular Ca2+ levels ([Ca2+]i) and force in vascular smooth muscle was mediated through activation of the thromboxane A2 and/or prostaglandin receptor, and (b) the relative roles of Ca2+ influx via L-type and non-L-type Ca2+ channels in prostaglandin receptor-mediated contraction. [Ca2+]i and force were measured simultaneously in fura-2-loaded rat aortic strips. The thromboxane A2 receptor antagonist, SQ29548 ([1S]-1a,2b(5Z),3b,4a-7-(3-[2-[(phenylamino)carbonyl] hydrazinomethyl)-7-oxobicyclo-[2.2.1]hept-2-yl-5-heptenoic acid), prevented the prostaglandin F2alpha-induced plateau [Ca2+]i elevation and force by 80-90%, while abolishing these responses due to the thromboxane A2 receptor agonist, U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F2alpha). Prostaglandin F2alpha (+ SQ29548)-induced plateau [Ca2+]i elevation and force were not inhibited by verapamil. Ni2+, a non-selective cation channel blocker, in the presence of verapamil, abolished the prostaglandin F2alpha (+ SQ29548)-elevated [Ca2+]i, while the contraction was only partially inhibited. These results suggest that, in rat aorta, (1) elevated [Ca2+]i and force due to high prostaglandin F2alpha concentrations largely results from thromboxane A2 receptor activation, and (2) the prostaglandin component of the prostaglandin F2alpha-induced contraction is dependent on Ca2+ influx via non-L-type channels.     

Histamine-induced contraction of human isolated bronchus is enhanced by endogenous prostaglandin F2 alpha and activation of TP receptors

The effect of histamine on the production of prostaglandin F2 alpha and the actions of prostaglandin F2 alpha on the responsiveness of human isolated bronchial smooth muscle were examined by organ bath techniques using bronchi from lung tissue resected from 18 patients. Following exposure to histamine, epithelium-intact bronchi generated 34.26 +/- 16.3 pg of prostaglandin F2 alpha/mg of tissue and epithelium-denuded preparations produced 32.62 +/- 11.83 pg/mg, suggesting that histamine-induced release of prostaglandin F2 alpha was from non-epithelial sources, presumably smooth muscle. The histamine H2 receptor antagonist ranitidine did not affect the release of prostaglandin F2 alpha, suggesting that its generation may have resulted from histamine H1 receptor activation. Carbachol did not influence prostaglandin F2 alpha generation. Contractile responses to histamine, prostaglandin F2 alpha and carbachol were measured in the presence and absence of the prostaglandin TP receptor antagonist SQ 29,548 ([1 S-[1 alpha,2 beta(5Z),3 beta,4 alpha]]-7-[3[[2-[9-phenylamino)carbonyl]hydrazino] methyl-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) (0.4 microM). SQ 29,548 abolished responses to prostaglandin F2 alpha suggesting that contractions were mediated via TP receptors. Exposure to SQ 29,548 also produced a 3-fold rightward shift in the concentration-effect curve for histamine (P = 0.01) without influencing the maximum response. SQ 29,548 did not affect responses to carbachol. These results suggest that histamine selectively stimulates the generation of prostaglandin F2 alpha from epithelium-denuded human airway tissue (presumably from the smooth muscle), which in turn, amplifies the contractile responses of human airway smooth muscle to histamine.     

Attenuation of Fos-like immunoreactivity in the trigeminal nucleus caudalis following trigeminovascular activation in the anaesthetised guinea-pig

The present study has examined the involvement of sensory neurotransmitters in activating neurones in the trigeminal nucleus caudalis following stimulation of the trigeminovascular system in anaesthetised guinea-pigs. Electrical stimulation of the right trigeminal ganglion produced a unilateral expression of Fos-like immunoreactivity (Fos-LI) in the trigeminal nucleus caudalis. The tachykinin NK1 receptor antagonist, GR205171 (100 micrograms/kg i.v.) and the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (1 mg/kg i.v.) each inhibited expression of Fos-LI following electrical stimulation. The calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37 (1.3 mg/kg i.v.), administered following rostral intracarotid infusion of mannitol to disrupt the blood-brain barrier, tended to reduce Fos-LI evoked by electrical stimulation, although this failed to reach statistical significance. Capsaicin (10 nmol in 0.1 ml), administered intracisternally, produced a bilateral expression of Fos-LI in the trigeminal nucleus caudalis. This expression was unaffected by the peripherally acting NK1 receptor antagonist, GR82334 (0.2 mg/kg i.v.) or CGRP8-37 (1.3 mg/kg i.v.). The centrally penetrant NK1 receptor antagonist, GR205171 (100 micrograms/kg i.v.), inhibited significantly Fos-LI evoked by intracisternal capsaicin administration. It is concluded that the sensory neurotransmitters, substance P and glutamate are released centrally following activation of the trigeminovascular system and that each may be involved in activation of cells in the trigeminal nucleus caudalis.     

Acetylcholine-induced endothelium-independent relaxations in monkey isolated superior and inferior caval veins

1. We examined the effects of acetylcholine (ACh), isoprenaline (Isop) and Ca-ionophore, A23187 on monkey isolated superior (SCV) and inferior caval veins (ICV) with and without intact endothelium, which had been partially contracted by 2 x 10(-6)-5 x 10(-6) M prostaglandin F2 alpha (PGF2 alpha). 2. Low concentrations of ACh (10(-10)-10(-9) M) produced a dose-dependent relaxation in the precontracted venous segments with endothelium. ACh at concentrations more than 10(-7) M elicited a transient contraction followed by a relaxation in these segments. 3. An addition of 5 x 10(-7) M A 23187 induced about 60% of maximum relaxation produced by 10(-5) M sodium nitroprusside (SNP) in each venous segment with endothelium. 4. Isop (10(-10)-10(-5) M) caused a dose-related relaxation in the precontracted caval veins with intact endothelium. 5. Removal of endothelium caused no significant effect on the ACh-induced dual responses but a significant inhibition of the A23187-induced relaxation. 6. Pretreatment with atropine antagonized competitively the ACh-induced relaxations in the endothelium-intact and endothelium-denuded caval veins. The Schild plot analysis showed that the pA2 values of the segments with and without endothelium were 9.72 +/- 0.14 (n = 5) and 10.01 +/- 0.23 (n = 6) in the ICV; and 9.95 +/- 0.20 (n = 5) and 9.70 +/- 0.10 (n = 5) in the SCV, respectively. 7. Pretreatment with 5 x 10-5M aspirin, 3 x 10-5M N0-nitro-L-arginine methylester, 1 mM tetraethylammonium,or 3 x 10-6 M glibenclamide caused no significant effect on the basal tone, ACh induced transient contraction, and ACh;.induced relaxation in the precontracted venous segments with and without endothelium.8. Pretreatment with 10-5 M methylene blue produced a significant reduction of the ACh- and SNP induced relaxations in the precontracted venous segments with and without endothelium. The pretreatment with the same concentration of methylene blue, however, caused no significant effect on the Isop-induced relaxation in venous segments with endothelium.9. The results suggest that ACh acts directly on the venous smooth muscle cells via a high-affinity muscarinic receptor subtype to accumulate cellular cyclic GMP producing endothelium-independent relaxation in the monkey caval veins.     

Uterine relaxation responses to calcitonin gene-related peptide and calcitonin gene-related peptide receptors decreased during labor in rats

OBJECTIVES: Our purpose was to investigate (1) whether uterine relaxation responses to calcitonin gene-related peptide are differentially regulated during pregnancy and labor, (2) the involvement of nitric oxide in smooth muscle relaxant action of calcitonin gene-related peptide in the rat uterus, (3) whether receptors for calcitonin gene-related peptide are expressed in rat uterus, and if so (4) whether the concentrations of these receptors are differently regulated during pregnancy and labor. STUDY DESIGN: Rats were killed on day 18 of gestation, at the time of spontaneous labor, or postpartum day 2. The uteri were removed for in vitro contractility measurements, nitric oxide production, and calcitonin gene-related peptide receptor binding assay. RESULTS: (1) Calcitonin gene-related peptide induced a dose-dependent relaxation in spontaneously contracting uterine strips from pregnant rats on day 18 of gestation; (2) the relaxation effects of calcitonin gene-related peptide on the uterus were decreased during spontaneous delivery at term and post partum compared with that during pregnancy; (3) calcitonin gene-related peptide-induced relaxation was inhibited by pretreatment of the uterine tissue with a calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide(8-37); (4) nitric oxide synthesis inhibitor (N(G)-nitro-L-arginine methyl ester) and soluble guanylate cyclase inhibitor (LY83583) significantly decreased calcitonin gene-related peptide-induced relaxation of the rat uterus during pregnancy; (5) calcitonin gene-related peptide increased the uterine nitric oxide production in pregnant rats, and this increase was obliterated in the presence of calcitonin gene-related peptide(8-37); and (6) calcitonin gene-related peptide receptors are present in rat uterus, and the concentration of these receptors dramatically increases during pregnancy and decreases during labor at term. CONCLUSIONS: Calcitonin gene-related peptide inhibits uterine spontaneous contractions in rats during pregnancy but not during labor and post partum. The inhibitory effects of calcitonin gene-related peptide on uterine contractility appear to be modulated, at least in part, by the activation of nitric oxide generation in the rat uterus. Changes in calcitonin gene-related peptide receptors could contribute to the changes in calcitonin gene-related peptide-mediated uterine relaxation during pregnancy and labor.     

Effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate in rats and mice

8-epi-prostaglandin F2 alpha stimulated contraction of human myometrial strips obtained from five different donors at the time of hysterectomy with a pEC50 value of 6.3 +/- 0.5. In paired strips from the same donors the pEC50 value for the selective TP receptor agonist U46619 ([1R-[1a,4a,5b(Z),6a(1E,3S*)]]-7-[6-(3- hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid) was 8.3 +/- 0.4. In strips from four other donors 8-epi-prostaglandin F2 alpha was ineffective whereas the pEC50 for U46619 was 6.9 +/- 0.3. Responses to 8-epi-prostaglandin F2 alpha were unaffected by the selective DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2- cyclohexyl-2-hydroxyethylamino)hydantoin) at 50 nM but were blocked by the selective TP receptor antagonist L670596 ((-)6,8-difluoro-9-p-methylsulfonyl benzyl-1,2,3,4- tetrahydrocarbazol-1-yl-acetic acid) at 50 nM. The pIC50 values obtained when the TP receptor antagonists GR 32191 ([1R- [1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'-biphenyl)-4- yl]methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), ICI D1542 ((4(Z)-6-[(2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]- 4-(3-pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid), ICI 192605 (4(Z)-6-[(2,4,5-cis)-2-(2-chlorophenyl)-4-(2-hydroxyphenyl)-1,3- dioxan-5-yl]hexenoic acid), L670596 and SQ 29548 ([1S-(1 alpha,2 beta(5Z),3 beta,4 alpha]]-7- [3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7- oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) were added cumulatively to strips pre-contracted with an EC80 concentration of 8-epi-prostaglandin F2 alpha were not significantly different from those obtained when an EC80 concentration of U46619 was used. The effects of 8-epi-prostaglandin F2 alpha on strips pre-contracted with an EC80 concentration of U46619 were not different from those of U46619 itself. It is concluded that in the non-pregnant human myometrium 8-epi-prostaglandin F2 alpha is a medium potency contractile agonist acting predominantly at the TP receptor.     

Endothelin-1 affects capsaicin-evoked release of neuropeptides from rat vas deferens

Capsaicin-sensitive neurones release a number of neuropeptides, such as substance P, neurokinin A, somatostatin and calcitonin gene-related peptide (CGRP), which exert a number of effects on smooth muscle tissues. Endothelin-1 was thought to potentiate the capsaicin-evoked release of neuropeptides from sensory neurones of the rat. We have investigated the neuromodulatory effects of endothelin-1 on capsaicin-induced release of neurotransmitters from rat vas deferens. Capsaicin and human alpha calcitonin gene-related peptide (human alphaCGRP) reduced the rat vas deferens twitch responses induced by electrical field stimulation. Human beta calcitonin gene-related peptide-(8-37) [human betaCGRP-(8-37)] (1 microM), a selective alphaCGRP receptor antagonist, antagonized the inhibitory effects of both drugs. Endothelin-1 concentration dependently evoked an increase in basal tone of the musculature and potentiated the amplitude of the electrically stimulated responses, blocking inhibitory effects of capsaicin but not of human alphaCGRP. Moreover, endothelin-1 did not markedly change the inhibitory effects of papaverine (0.1-100 microM) or isoprenaline (1 nM-100 microM) on responses to electrical field stimulation. FR 139317 [(N,N-hexamethylene) carbamoyl-Leu-D-Trp(N-Me)-D-2-Pya], a selective endothelin ET(A) receptor antagonist, administered 30 min before endothelin-1 restored the capsaicin effects whereas BQ 788 [Dmpc-gamma-MeLeu-D-Trp-(1-methoxycarbonyl)-D-Nle], a selective endothelin ET(B) receptor antagonist, was completely ineffective. The endothelin-1-induced block of the capsaicin effect was resistant to tetrodotoxin (1 microM) and 30-min pre-treatment with MEN 10.627 (cyclo[(Met-Asp-Trp-Phe-Dap-Leu) cyclo (2beta-5beta)]), a selective tachykinin NK2 receptor antagonist, did not abolish the endothelin-1 effect on the inhibitory response to capsaicin. These results suggest that endothelin-1 selectively inhibits the capsaicin-induced release of neurotransmitters from rat vas deferens and these effects are mediated via endothelin ET(A) receptors but not by tachykinin release.     

Tachykinin receptors in the small intestine of the cane toad (Bufo marinus): a radioligand binding and functional study

In this study, we have used radioligand binding and functional techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [125I]Bolton-Hunter substance P ([125I]BHSP) was saturable, of high affinity (Kd 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin approximately neurokinin A (NKA) > or = SP(5-11) > or = neuropeptide gamma > or = scyliorhinin II > scyliorhinin I > or = [Sar9]-SP > or = neurokinin B approximately physalaemin approximately carassin > SP(7-11) approximately eledoisin > or = SP(4-11) approximately SP(6-11). Binding was also inhibited by Gpp[NH]p > or = GTPgammaS > App[NH]p, indicating a G-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC50 1.4 nM) > eledoisin approximately SP > or = physalaemin > or = ranakinin > SP(6-11) > scyliorhinin II > or = neuropeptide gamma > neurokinin B approximately NKA approximately scyliorhinin I > or = SP(4-11) > or = SP(5-11) > [Sar9]SP > SP(7-11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar9,Met(O2)11]SP, [Lys5,Me-Leu9,Nle10]NKA(4-10) and senktide were weak or ineffective. There was a strong positive correlation between the pD2 and pIC50 values for mammalian tachykinins and analogues (r = 0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar9,Met(O2)11]-SP(pD2 5.7) was approximately 25-fold less potent as an agonist than [Sar9]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP (spantide; 1 microM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 microM) were ineffective in both functional and binding studies. Tetrodotoxin (1 microM) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [125I]BHSP prefers SP and ranakinin. This toad "NK-1-like receptor" differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.     

Genetic construction and characterization of an anti-monkey CD3 single-chain immunotoxin with a truncated diphtheria toxin

In arteries, adrenomedullin (ADM) causes relaxations of rings with and without endothelium by stimulating accumulation of cyclic nucleotides resulting from activation of the ADM and calcitonin gene-related peptide (CGRP) receptors. Experiments were designed to determine the mechanism(s) of relaxation to ADM in veins. Rings of canine femoral vein with and without endothelium were suspended in organ chambers for measurement of isometric force. Rings were contracted with prostaglandin F2alpha (2 x 10(-6) M), and cumulative dose-responses to ADM (10(-11) to 10(-7) M) were obtained in the absence or presence of indomethacin (10(-5) M), indomethacin + N(G)-monomethyl-L-arginine (10(-4) M), methylene blue (10(-5) M), particulate guanylate cyclase inhibitor HS-142-1 (10(-5) M), tetraethylammonium (TEA, 10(-2) M), CGRP-receptor antagonist (CGRP 8-37, 10(-6) M), ADM-receptor antagonist (ADM 26-52, 10(-6) M), diphenhydramine (10(-6) M), 8-phenyltheophylline (3 x 10(-6) M), or superoxide dismutase (150 U/ml) plus catalase (1,200 U/ml). ADM produced concentration-dependent relaxations only in veins with endothelium. Relaxations to ADM in rings with endothelium were significantly inhibited only by methylene blue and HS-142-1. In separate experiments, incubation of rings with ADM (10(-8) M) and 3-isobutyl-1-methyl-xanthine (10(-4) M) for 3 min did not significantly affect the accumulation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). These data suggest that ADM-mediated relaxation in veins is endothelium dependent and is not associated with activation of CGRP receptors or currently defined ADM receptors. Further, relaxations are not mediated by nitric oxide, indomethacin-sensitive prostanoids, TEA-sensitive hyperpolarizing factors, oxygen free radicals, or accumulation of cyclic nucleotides.     

Vasorelaxation in isolated bone arteries. Vasoactive intestinal peptide, substance P, calcitonin gene-related peptide, and bradykinin studied in pigs

We assessed the effects of vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), and bradykinin in arteries (diameter approximately 230 microns) isolated from cancellous bone from pigs. Arterial segments (2 mm long) were mounted on a myograph for measurement of isometric force development. After submaximal precontraction with norepinephrine, VIP (10(-10)-10(-7) M), CGRP (10(-11)-10(-7) M), SP (10(-6) M), and bradykinin (10(-11)-10(-6) M) were added. 44 arterial segments (23 pigs) were investigated. VIP-, CGRP-, and bradykinin induced a concentration-dependent vasorelaxation, while SP mediated a transient relaxation. After mechanical removal of the endothelium, the effects of SP and bradykinin were completely abolished, while the relaxation to CGRP was still pronounced. This indicates that the effects of SP and bradykinin are mediated by the endothelium, while CGRP mainly mediates relaxation by a direct effect on vascular smooth muscle cells. The relaxations to CGRP and bradykinin were still significant after inhibition of nitric oxide synthase with 10(-4) M N omega-nitro-L-arginine (L-NNA) and inhibition of prostaglandin synthesis with 10(-5) M indomethacin, indicating the existence of an alternative vasorelaxing pathway. Our findings support the theory of a vasoregulatory role of neuropeptides in bone.     

Endothelium-dependent potentiation of prostaglandin F2alpha-induced contractions by (+/-)-[6]-gingerol is inhibited by cyclooxygenase- but not lipoxygenase-inhibitors in mouse mesenteric veins

The mechanism of potentiation of prostaglandin (PG) F2alpha-induced contraction of mouse mesenteric veins by (+/-)-[6-gingerol was investigated in vitro. (+/-)-[6]-Gingerol (0.3mM) potentiated the maximal contraction response elicited by PGF2alpha (0.28 mm) in the presence of intact vascular endothelium, but not in its absence (de-endothelialized preparations). The potentiating effect was completely inhibited by cyclooxygenase inhibitors (0.2 mm aspirin and 0.2 mm indomethacin) and partly by calcium antagonists (2 microM verapamil, 8 nM nitrendipine and 1 microM ryanodine), but not inhibited by nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor and ONO-3708, a thromboxane (TX) A2 antagonist. The potentiation by (+/-)-[6]-gingerol is also observed in mesenteric veins of streptozotocin-diabetic mice where the enhancement of PGF2alpha-induced contraction is caused mainly by activation of lipoxygenase. The potentiation of PGF2alpha-induced contraction by (+/-)-[6]-gingerol may be caused by a cyclooxygenase-dependent release of vasoconstrictors, other than PGF2alpha and TXA2, or by inhibiting vasorelaxants released from endothelial cells of mouse mesenteric veins.     

The tachykinin NK1 receptor antagonist, RP67580, inhibits the bradykinin-induced rise in intracellular Ca2+ concentration in bovine pulmonary artery endothelial cells

The bradykinin-induced rise in intracellular Ca2+ concentration ([Ca2+]i) and the bradykinin receptor involved in this response were characterized in bovine pulmonary artery endothelial cells. It was found that bradykinin induces an intracellular biphasic Ca2+ response, consisting of a transient peak followed by an elevated plateau phase. Both bradykinin and the bradykinin B1 receptor agonist, des-Arg9-bradykinin, induced a concentration-dependent increase in [Ca2+]i, but the bradykinin-induced rise was much greater. Moreover, the bradykinin-induced [Ca2+]i rise could be inhibited by the bradykinin B2 receptor antagonists, D-Arg0[Hyp3, Thi(5,8), D-Phe7]bradykinin and Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]bradykinin), but not by the bradykinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin. From these results it can be concluded that a bradykinin B2 receptor is involved in this response. Furthermore, we found that the tachykinin NK1 receptor antagonist, RP67580 ([imino 1 (methoxy-2-phenyl)-2 ethyl]-2 diphenyl 7,7 perhydroisoindolone-4 (3aR, 7aR)), and its negative enantiomer, RP68651 (2-[1-imino 2-(2 methoxy phenyl) ethyl] 7,7 diphenyl 4-perhydroisoindolone (3aS-7aS)), could inhibit the bradykinin-induced [Ca2+]i response, although no functional tachykinin NK1 receptors were found. Binding studies evidenced no binding of RP67580 or RP68651 to the bradykinin receptor. We conclude that RP67580 inhibits the bradykinin-induced rise in [Ca2+]i via a bradykinin B2 receptor-independent mechanism.     

Flufenamic and tolfenamic acids and lemakalim relax guinea-pig isolated trachea by different mechanisms

The effects of K+ channel inhibitors on the relaxations induced by flufenamic and tolfenamic acids and lemakalim were examined in guinea-pig isolated trachea precontracted with prostaglandin F2alpha (PGF2alpha, 1 microM). Flufenamic and tolfenamic acids (0.1-33 microM) and lemakalim (0.01-33 microM) relaxed guinea-pig trachea in a concentration-dependent manner. Tetraethylammonium (0.5-2 mM), a nonspecific inhibitor of K+ channels, inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Charybdotoxin (ChTX, 33-100 nM), an inhibitor of the large Ca2+-activated K+ channels (BK(Ca)), also inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Glipizide (3.3-33 microM), an inhibitor of the ATP-sensitive K+ channels (K(ATP)) inhibited lemakalim-induced relaxation without affecting those induced by flufenamic and tolfenamic acids. Our results indicate that the relaxations of guinea-pig isolated trachea by flufenamic and tolfenamic acids are due to activation of BK(Ca). The relaxant mechanism of flufenamic and tolfenamic acids thus differs from that of lemakalim, an activator of K(ATP).     

Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea-pig common bile duct

Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 microM), produced a slowly-developing contraction ('on' response) followed by a quick phasic 'off' contraction ('off peak' response) and a tonic response ('off late' response), averaging 16+/-2, 73+/-3 and 20+/-4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 microM; 15 min before) abolished the overall response to EFS (n 8). Neither in vitro capsaicin pretreatment (10 microM for 15 min), nor guanethidine (3 microM, 60 min before) affected the excitatory response to EFS (n 5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. Nomega-nitro-L-arginine (L-NOARG, 100 microM, 60 min before) or naloxone (10 microM, 30 min before) significantly enhanced the 'on' response (294+/-56 and 205+/-25% increase, respectively; n=6-8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655+/-90% increase of the 'on' component, n = 6, P<0.05). The tachykinin NK2 receptor-selective antagonist MEN 11420 (1 microM) almost abolished both the 'on' and 'off late' responses (P<0.01: n=5 each) to EFS, and reduced the 'off-peak' contraction by 55+/-8% (n=5, P<0.01). The subsequent administration of the tachykinin NK1 receptor-selective antagonist GR 82334 (1 microM) and of the tachykinin NK3 receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 microM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 microM, GR 82334 significantly reduced (by 68+/-9%, P<0.05, n=6) the 'on' response to EFS. The contractile 'off peak' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 microM each) was abolished (P<0.01; n=6) by the administration of the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM). PPADS (30 microM) selectively blocked (75+/-9 and 50+/-7% inhibition, n = 4 each) the contractile responses produced by 100 and 300 microM ATP. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK2 (and possibly NK1) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.     

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.     

Angiotensin II inhibits glomerular adenylate cyclase via the angiotensin II receptor subtype 1 (AT1)

We examined the role of angiotensin II (AII) receptor subtypes in the regulation of hormone-stimulated cyclic AMP (cAMP) accumulation in isolated rat glomeruli. All inhibited cAMP formation induced by histamine, serotonin and parathyroid hormone, but not by prostaglandin E2 or calcitonin gene-related peptide. Angiotensin III but not the angiotensin fragments (1-7) and (3-8) also showed inhibitory activity. The inhibition of histamine-induced cAMP accumulation by AII was concentration-dependent and was absent in glomeruli isolated from pertussis toxin-treated rats. The effect of AII on histamine-induced cAMP levels was not mimicked by the protein kinase C activator, phorbol-12-myristate-13-acetate, nor was the effect of AII inhibited by the protein kinase C inhibitors, staurosporine and H-7. The angiotensin II receptor subtype 1 (AT1) antagonists, SK&F 108566 and losartan, attenuated the inhibitory effect of AII on histamine-stimulated cAMP accumulation, whereas the AT2 selective antagonists, CGP 42112A, WL-19 and PD 123319, had no effect. Displacement of [125I]AII from glomerular membrane using the subtype-selective antagonists confirmed that the glomerular AII receptor has characteristics of an AT1 subtype. The results suggest that AII, through activation of the AT1 receptor, may act to maintain the contractile state of glomerular mesangial cells by attenuating the increase in cAMP levels induced by some hormones.     

Monkey central retinal artery is innervated by nitroxidergic vasodilator nerves

PURPOSE: To determine whether the monkey central retinal artery is innervated by vasodilator nerves and to analyze the mechanism underlying the neurogenic response. METHODS: Changes in isometric tension were recorded in helical strips of the arteries, which were stimulated by transmurally applied electrical pulses or nicotine. The presence of perivascular nerve fibers containing nitric oxide (NO) synthase immunoreactivity was determined histologically. RESULTS: Transmural electrical stimulation (5 Hz) and nicotine produced a relaxation of the arterial strips denuded of the endothelium, treated with prazosin, and contracted with prostaglandin F2 alpha. The response was not influenced by timolol, atropine, and indomethacin, but it was abolished by methylene blue and oxyhemoglobin. NG-nitro-L-arginine, a NO synthase inhibitor, abolished the neurogenic relaxation, and L-arginine restored the response. Antagonists of calcitonin gene-related peptide and vasoactive intestinal polypeptide in sufficient concentrations did not influence the response to nerve stimulation by nicotine. There were abundant nerve fibers and bundles containing NO synthase immunoreactivity in the adventitia. CONCLUSIONS: Monkey central retinal arteries are innervated by NO synthase-containing nerves that liberate NO possibly as a neurotransmitter on excitation to produce muscular relaxation.     

Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon

1. The effect of tachykinin NK1 and NK2 receptor antagonists on noncholinergic excitatory junction potentials (e.j.ps) evoked by electric field stimulation (EFS) in the circular muscle of the guinea-pig proximal colon was investigated by means of a sucrose-gap technique. 2. In the presence of 1 microM atropine, submaximal EFS (10 Hz, 20-30 V, 0.5 ms pulse width, 1 s train duration) evoked an inhibitory junction potential (i.j.p.) followed by e.j.p. with superimposed action potentials (APs) and contraction. Addition of either NG-nitro-L-arginine (L-NOARG, 0.1 mM) or apamin (0.1 microM) inhibited the evoked i.j.p. and the combined administration of the two agents almost abolished it. In the presence of both L-NOARG and apamin, an atropine-resistant e.j.p. was the only electrical response evoked by EFS in 50% of cases and a small i.j.p. (10% of original amplitude) followed by e.j.p. was evident in the remainder. 3. In the presence of L-NOARG and apamin, the tachykinin NK1 receptor antagonists, (+/-)-CP 96,345 and GR 82,334 (10 nM-3 microM) concentration-dependently inhibited the atropine-resistant e.j.p. and accompanying contraction evoked by EFS. EC50 values were: 0.77 microM (e.j.p. inhibition) and 0.22 microM (inhibition of contraction) for (+/-)-CP 96,345; 0.61 microM (e.j.p. inhibition) and 0.20 microM (inhibition of contraction) for GR 82,334. The tachykinin NK2 receptor antagonists, MEN 10,376 (up to 3 microM) and SR 48,968 (up to 1 microM) had no effect on the atropine-resistant e.j.p. MEN 10,376 (3 microM) but not SR 48,968 produced a slight inhibition of the evoked contraction. 4. (+/- )-CP 96,345 (3 microM) and GR 82,334 (3 microM) markedly reduced (81 and 89% inhibition, respectively)the atropine-resistant ej.p. in the absence of L-NOARG and apamin, without affecting the ij.p. MEN 10,376 (3 microM) and SR 48,968 (1 microM) had no significant effect on noncholinergic ij.p. and ej.p. evoked in the absence of apamin and L-NOARG.5. The electrical and mechanical responses to the NK, receptor agonist [Sar9]substance P (SP) sulfone were blocked by (+/-)-CP 96,345 (3 1M) or GR 82,334 (3 microM) which, at the same concentration, failed to affect the responses to the NK2 receptor agonist [PAla8] neurokinin A (NKA) (4-10). In contrast, MEN10,376 (3 microM) or SR 48,968 (1 microM) blocked the response to [beta Ala8]NKA(4-10) without affecting the response to [Sar9]SP sulfone.6. In the presence of L-NOARG and apamin, and in the absence of atropine, EFS of low pulse width(0.02-0.03 ms, other parameters as above) produced cholinergic ej.ps and contraction which were unaffected by GR 82,334 (3 microM). (+/-)-CP 96,345 (3 JAM) produced 24% reduction in the area of the atropine-sensitive ej.p. without affecting the peak amplitude of ej.p. or contraction.7. These findings demonstrate that the noncholinergic ej.ps and accompanying contraction of the circular muscle of the guinea-pig colon are produced through activation of intramural tachykininergic nerves and that the resultant smooth muscle response is almost entirely mediated through NK1 receptors.     

The F2-isoprostane 8-epiprostaglandin F2alpha increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.     

Mechanisms of relaxation of coronary artery by hypoxia

This study was designed to clarify the dependency of hypoxic coronary vasodilation (HCD) on the endothelium and the role of the K+ channels on HCD in the rabbit coronary artery. HCD was investigated in an isolated left circumflex coronary artery precontracted with prostaglandin F2 alpha. Vascular rings were suspended for isometric tension recording in an organ chamber filled with Krebs-Henseleit (KH) solution. Hypoxia was induced by gassing the chamber with 95% N2 + 5% CO2 and was maintained for 15 approximately 25 min. Hypoxia elicited a vasodilation in the precontracted coronary artery with and without endothelium. There was no difference between the amplitude of the HCD induced by two consecutive hypoxic challenges and the effects of 20% O2 + 5% CO2 + 75% N2 and 95% O2 + 5% CO2 control K-H solution of subsequent responses to hypoxia. Inhibition of the cyclooxygenase pathway by treatment with indomethacin had no effect on HCD. Blockades of the tetraethylammonium chloride-sensitive K+ channel abolished HCD. Apamin, a blocker of the small conductance Ca(2+)-activated K+ (KCa) channel, and iberiotoxin, a blocker of the large conductance KCa channel had no effect on HCD, respectively. Glibenclamide, a blocker of the ATP-sensitive K+ (K+ATP) channel, reduced HCD. Cromakalim, an opener of the K+ATP channel, relaxed the coronary artery precontracted with prostaglandin F2 alpha. The degree of relaxation by cromakalim was similar to that by hypoxia while glibenclamide reduced both hypoxia- and cromakalim-induced vasodilatations. In conclusion, these results suggest that HCD is independent on endothelium and HCD is considered to be induced by activation of K+ATP channel.