Alpha2-adrenoceptor-mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP-dependent and a cyclic AMP-independent mechanism

1. The aim of this study was to determine the conditions under which the alpha2-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2. UK14304 (0.3 microM) produced a small contraction of porcine isolated ear arteries which was 7.8+/-3.3% of the response to 60 mM KCl. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 microM U46619 (8.2+/-1.8% and 10.2+/-2.6% of 60 mM KCl response, respectively). However, an enhanced alpha2-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of UK14304 (46.9+/-9.6% of 60 mM KCl response). 3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the alpha1-adrenoceptor antagonist prazosin (0.1 microM), but were inhibited by the alpha2-adrenoceptor antagonist rauwolscine (1 microM), indicating that the enhanced responses were mediated via postjunctional alpha2-adrenoceptors. 4. In the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 microM forskolin produced an increase in [3H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 microM UK14304 prevented this increase. 5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9+/-9.6% of the 60 mM KCl response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8+3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2+/-1.7%, which was not different from the control response in the same tissues (12.2+/-5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2+/-1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7. In conclusion, enhanced contractions to the alpha2-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.     

The arterial blood supply of the human patella. Its clinical importance for the operating technique in vascularized knee joint transplantations

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK1 and NK2 receptor agonists, [Sar9]substance P (SP) sulfone and [beta Ala8]neurokinin A (NKA) (4-10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 microM) and indomethacin (10 microM). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nM) was selected: [Sar9]SP sulfone (10 nM) produced a biphasic contraction, the two amplitudes averaging 75 +/- 2 and 43 +/- 3% of the maximal response to KCl (80 mM) at 1 and 15 min from application of the agonist, respectively. CPA (3 microM for 60 min) slightly reduced the phasic response to [Sar9]SP sulfone (16 +/- 4% inhibition) and markedly suppressed the tonic component (89 +/- 3% inhibition). 3 The contraction produced by [beta Ala8]NKA (4-10) (10 nM) was more sustained than that induced by the NK1 receptor agonist: it averaged 69 +/- 5 and 73 +/- 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [beta Ala8]NKA (4-10) (18 +/- 7 and 21 +/- 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK1 and NK2 receptor antagonists (SR 140333 and MEN 10627, respectively, 1 microM each) and of L-nitroarginine (100 microM), KCl (40 mM) produced a distinct phasic and tonic contraction which was suppressed by 1 mM nifedipine. CPA (3 microM) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 microM nifedipine, the response to [beta Ala8]NKA (4-10) was slightly depressed (32 +/- 6% inhibition) in its early component only, while the response to [Sar9]SP sulfone was abolished. CPA produced a slight inhibition (15 +/- 9 and 33 +/- 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [beta Ala8]NKA (4-10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [beta Ala8]NKA (4-10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar9]SP sulfone (0.1 microM for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 microM) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar9]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK1 receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK2 receptor-mediated contractile responses.     

Developmental toxicity study of inhaled acrylic acid in New Zealand White rabbits

The effect of Rp diastereoisomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) on relaxation elicited by histamine (1-100 microM), forskolin (1-60 microM), papaverine (1-100 microM), vinpocetine (1-100 microM), rolipram (0.1-1 mM), Sp-cAMPS (10-300 microM), 8-BrcAMP (10 microM - 1 mM) and 8-BrcGMP (3 microM - 1 mM) of the previous vanadate-induced contraction was assayed. The effect of Rp-cAMPS on the relaxing effect produced by forskolin, papaverine, vinpocetine, rolipram, Sp-cAMPS and 8-BrcAMP in KCl-induced tonic contraction was also assayed. Histamine, forskolin, papaverine, rolipram, Sp-cAMPS, 8-BrcAMP and 8-BrcGMP, but not vinpocetine, relaxed the vanadate-induced contractions in rat uterus incubated in medium lacking calcium plus EDTA in a concentration-dependent way. Rp-cAMPS (1-300 microM) had no effect on vanadate contraction. However, it antagonized the relaxation elicited by histamine and papaverine, but not that of forskolin, rolipram, Sp-cAMPS, 8-BrcAMP and 8-BrcGMP. Forskolin, papaverine, vinpocetine, rolipram and 8-BrcAMP, but not Sp-cAMPS, relaxed the KCl-induced contraction. Rp-cAMPS antagonized the relaxation elicited by forskolin, papaverine and vinpocetine, but not that of rolipram and 8-BrcAMP. Our results suggest that: a) Rp-cAMPS is an effective PKA inhibitor that could be used to study the involvement of cAMP on drug-induced response in smooth muscle, and b) the effects of Sp-cAMPS, 8-BrcAMP and rolipram were independent of the activation of protein kinases.     

Genistein inhibits slow component delayed-rectifier K currents via a tyrosine kinase-independent pathway

In single guinea pig ventricular cells, genistein, a potent inhibitor of protein tyrosine kinase (PTK), was found to suppress the delayed-rectifier K (IK) current. The present study was carried out to examine the underlying mechanism. Ventricular myocytes were voltage-clamped in the conventional whole-cell mode (36 degrees C). The amplitudes of tail and steady-state (2-s pulse) currents were measured as IK. Genistein (10-100 microM) reversibly inhibited both basal and intrapipette cAMP (1 mM)-enhanced IK currents in a concentration-dependent manner with a half-maximum inhibitory concentration (IC50) at approximately 30 microM. In contrast, lavendustin A (10 microM; n = 5) and tyrphostin 51 (100 microM; n = 5) had no effect on the currents. The inhibitory action of genistein was also seen after IK currents were activated by forskolin (500 nM) plus intrapipette orthovanadate (500 microM). The intrapipette cAMP-enhanced IK was also reduced to a lesser degree by daidzein, an inactive analogue of genistein. Envelope tail and short pulse protocols revealed that genistein inhibits the slow component of IK (IKs). Thus, the inhibitory action of genistein is not mediated via an inhibition of PTK but may be due to the block of IKs channels.     

Nitric oxide, a possible mediator of 1,4-dihydropyridine-induced photorelaxation of vascular smooth muscle

1. In rat aortic tissues pre-contracted with phenylephrine, certain 1,4-dihydropyridines (DHPs) such as Bay K 8644 (0.1 microM), PN 202791 (1 microM), RK 30 (1 microM), NI 104 (1 microM) and NI 105 (1 microM) enhanced photoactivated relaxations (photorelaxation or PR) whereas NI 72, NI 85, NI 99, NI 102, amlodipine, felodipine, nifedipine and nimodipine were inactive. 2. The PR inducing effects of Bay K 8644 were mimicked by the diabetogenic agent, streptozotocin (STZ). 3. Solutions of Bay K 8644 which had been irradiated for various periods of time initiated light independent transient relaxations followed by contractile responses in aortic tissue partially contracted with phenylephrine. With exposure times to light of 30 to 120 min, the intensity of the relaxation response to irradiated Bay K 8644 increased from 26 +/- 3.3 to 71 +/- 3.7% of the maximum contractile response to phenylephrine (n = 5). Conversely the contractile responses decreased, from 84.2 +/- 4.1 to 19.8 +/- 10.4% of the maximum contractile response to phenylephrine (n = 5). 4. Superoxide ions, generated by incubation of xanthine (2mM) plus xanthine oxidase (10 mu ml-1) in physiological saline solution (PSS) NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 12.5 and glucose 11.1 (mM) for 1 h. reduced the PR induced by DHPs, STZ, and also NO-induced relaxations of rat aortic preparations. 5. Direct measurements of NO indicate that, following exposure to a polychromatic light source, equimolar concentrations (0.1 mM) of the DHP compounds that enhance PR, as well as STZ, photodegrade to release NO (25 +/- 2-40.3 +/- 5.9 nmol min-1, n = 6). 6. Structure-activity studies indicate that a nitro group at the -3 position of the dihydropyridine ring is essential for DHPs to support PR. 7. These data suggest that the photodegradation of DHPs and STZ leading to the release of NO provides the primary cellular process underlying the PR response.     

The ETA antagonist CI-1020 inhibits hypoxic pulmonary vasoconstriction in small isolated rat pulmonary arteries

Hypoxic pulmonary vasoconstriction (HPV) of rat pulmonary arteries in vitro occurs in four phases. Initial vasodilation (phase 1), is followed by transient contraction (phase 2), further vasodilation (phase 3) and finally a second sustained contraction (phase 4). We have investigated the role of ET-1 in HPV using the ETA receptor antagonist CI-1020. Small rat pulmonary arteries (SPA, n=32, diameter=454+/-22 microM) were mounted in a wire myograph. Two contractions to 80 microM KCl ensured response reproducibility and relaxation to 10 microM acetyl choline following constriction with 100 microM prostaglandin F2alpha (PGF2alpha) to indicate endothelial integrity. A control hypoxic response was produced following priming with 5 microM PGF2alpha. Vessels (n=8) were then exposed to either vehicle or CI-1020 (1, 10 or 100 microM) for 30 min in the dark before re-exposure to PGF2alpha and hypoxia. Responses were standardized as a percentage of contraction to 80 mM KCl. Vehicle caused an increase in phase 2 of HPV of +2.51+/-4.20% (expressed as difference between pre- and post-drug values). CI-1020 (1, 10 and 100 microM) caused a significant reduction in phase 2 of HPV of -9. 76+/-1.40%, -9.23+/-2.30% and -7.96+/-1.70%, respectively (P<0.05). These results suggest that phase 2 of HPV in rat SPA is attributed, in part, to the action of ET-1 at the ETA receptor.     

Effect of niflumic acid on noradrenaline-induced contractions of the rat aorta

1. The effects of niflumic acid, an inhibitor of calcium-activated chloride channels, were compared with the actions of the calcium channel antagonist nifedipine on noradrenaline-evoked contractions in isolated preparations of the rat aorta. 2. The cumulative concentration-effect curve to noradrenaline (NA) was depressed by both nifedipine and niflumic acid in a reversible and concentration-dependent manner. The degree of inhibition of the maximal contractile response to NA (1 microM) produced by 10 microM niflumic acid (38%) was similar to the effect of 1 microM nifedipine (39%). 3. Contractions to brief applications (30 s) of 1 microM NA were inhibited by 55% and 62% respectively by 10 microM niflumic acid and 1 microM nifedipine. 4. In the presence of 0.1 microM nifedipine, niflumic acid (10 microM) produced no further inhibition of the NA-evoked contractions. Thus, the actions of niflumic acid and nifedipine were not additive. 5. In Ca-free conditions the transient contraction induced by 1 microM NA was not inhibited by niflumic acid (10 microM) and therefore this agent does not reduce the amount of calcium released from the intracellular store or reduce the sensitivity of the contractile apparatus to calcium. 6. Niflumic acid 10 microM did not inhibit the contractions produced by KCl (up to 120 mM) which were totally blocked by nifedipine. Contractions induced by 25 mM KCl were completely inhibited by 1 microM levcromakalim but were unaffected by niflumic acid. 7. It was concluded that niflumic acid produces selective inhibition of a component of NA-evoked contraction which is probably mediated by voltage-gated calcium channels. These data are consistent with a model in which NA stimulates a calcium-activated chloride conductance which leads to the opening of voltage-gated calcium channels to produce contraction.     

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.     

Interaction between Ca2+, K+, carbamazepine and zonisamide on hippocampal extracellular glutamate monitored with a microdialysis electrode

1. Multiple components of hippocampal glutamate release were examined by study of Ca2+- and K+-evoked hippocampal extracellular glutamate release using an in vivo microdialysis glutamate biosensor in urethane-anaesthetized rats. In addition, the effects of the antiepileptic drugs, carbamazepine (CBZ) and zonisamide (ZNS) perfused through the probe on glutamate release were assessed. 2. Basal glutamate levels were below detection limits (approximately 0.1 microM). An increase in extracellular KCl (from 2.7 to 50 and 100 mM) increased extracellular hippocampal glutamate levels to 9.2+/-1.4 and 20.0+/-2.6 microM, respectively, calculated from the area under curve (AUC) for 60 min. 3. This KCl-evoked glutamate release consisted of three components: an initial transient rise, a late gentle rise, and late multiple phasic transient rises. 4. An increase in or removal of extracellular CaCl2 levels respectively enhanced and reduced the 50 mM KCl-evoked hippocampal glutamate release (AUC for 60 min) from 9.2+/-1.4 to 12.4+/-2.1 and 5.8+/-0.9 microM. 5. Perfusion with 100 microM CBZ or 1 mM ZNS inhibited both the 50 mM KCl-evoked hippocampal glutamate release (AUC for 60 min) from 9.2+/-1.4 to 5.5+/-1.1 and to 5.8+/-1.3 microM, respectively, as well as the stimulatory effects of Ca2+ on KCl-evoked hippocampal glutamate release. 6. These results suggest that both CBZ and ZNS may reduce epileptiform events by inhibiting excitatory glutamatergic transmission.     

Canine bronchial sustained contraction in Ca2+-free medium: role of intracellular Ca2+

We evaluated whether cartilage was a source of Ca2+ and the possible role of Ca2+ recycling in the sustained bronchial contraction (SBC) induced by carbachol (Cch) in Ca2+-free medium. Canine first-order bronchi were studied with cartilage and epithelium (+CAR + EPI) and without these structures individually (-CAR + EPI and +CAR - EPI) or together (-CAR - EPI). After cartilage removal (-CAR - EPI or -CAR + EPI) Cch produced a transient contraction in Ca2+-free medium. Removal of the epithelium alone had minor effects on the magnitude of the SBC but increased the effect of removal of cartilage to diminish the SBC. Bronchial strips with cartilage were able to respond to Cch with lower Ca2+ concentrations (10-100 microM) than could dissected preparations. Preincubation with BAY K 8644 (30-1000 nM) or 60 mM KCl or -CAR - EPI tissues converted the transient contractions to Cch in Ca2+-free medium to sustained contractions. In microelectrode studies, 50 nM Cch induced membrane oscillations in solutions with 2.5 mM Ca2+ in bronchial preparations, plus or minus cartilage, and in undissected tissues in Ca2+-free medium but not in -CAR - EPI tissues. Preincubation with 1 microM BAY K 8644 in Ca(2+)-free medium restored these oscillations in -CAR - EPI tissues. The release of 45Ca2+ from cartilage was too rapid to provide a reservoir of Ca2+ to support multiple SBCs in Ca2+-free medium. Moreover, in the Ca2+-free medium (with 10 nM Ca2+ after tissue +CAR + EPI incubation) excitatory junction potentials rapidly disappeared. Addition of 1 microM nifedipine or 1 mM EGTA during the SBC of +CAR + EPI tissues produced complete relaxation. A transient contraction to Cch occurred with prior addition of nifedipine. Inhibition of the sarcoplasmic reticulum Ca2+ pump by tissue incubation with cyclopiazonic acid (CPA; 10 microM), or briefly with 1 mM EGTA significantly diminished the SBC induced by Cch in Ca2+-free medium. CPA and EGTA together abolished the Cch-induced SBC. Thus, cartilage plays a more complex role than as a Ca2+ reservoir to support the SBC induced by Cch in Ca2+-free solution; its removal affects the process supporting SBCs involving intracellular Ca2+ storage and Ca2+ entrance through voltage-dependent channels.     

A method for isolating adult and neonatal airway smooth muscle cells and measuring shortening velocity

Methods are described for isolating smooth muscle cells from the tracheae of adult and neonatal sheep and measuring the single-cell shortening velocity. Isolated cells were elongated, Ca2+ tolerant, and contracted rapidly and substantially when exposed to cholinergic agonists, KCl, serotonin, or caffeine. Adult cells were longer and wider than preterm cells. Mean cell length in 1.6 mM CaCl2 was 194 +/- 57 (SD) microm (n = 66) for adult cells and 93 +/- 32 microm (n = 20) for preterm cells (P < 0.05). Mean cell width at the widest point of the adult cells was 8.2 +/- 1.8 microm (n = 66) and 5.2 +/- 1.5 microm (n = 20) for preterm cells (P < 0.05). Cells were loaded into a perfusion dish maintained at 35 degreesC and exposed to agonists, and contractions were videotaped. Cell lengths were measured from 30 video frames and plotted as a function of time. Nonlinear fitting of cell length to an exponential model gave shortening velocities faster than most of those reported for airway smooth muscle tissues. For a sample of 10 adult and 10 preterm cells stimulated with 100 microM carbachol, mean (+/- SD) shortening velocity of the preterm cells was not different from that of the adult cells (0.64 +/- 0.30 vs. 0.54 +/- 0.27 s-1, respectively), but preterm cells shortened more than adult cells (68 +/- 12 vs. 55 +/- 11% of starting length, respectively; P < 0.05). The preparative and analytic methods described here are widely applicable to other smooth muscles and will allow contraction to be studied quantitatively at the single-cell level.     

Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by alpha-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-induced NO synthesis. Metabolism of L-[3H]arginine to L-[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) or spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70+/-0.07%) by L-NMMA (100 microM). Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (approximately 2 fold) iNOS protein expression. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.     

Synergistic action of quercetin and genistein in human ovarian carcinoma cells

Ovarian carcinoma is the fourth most common cause of cancer death in women and there has been a steady increase in the age-adjusted cancer death rates in the past 25 years in the US. However, patients who become cisplatin resistant respond poorly to available cytotoxic agents; therefore, discovering novel targets for ovarian carcinoma is vital. Quercetin, an anticancer agent, arrests the cell cycle at G1 and S phase boundary. Genistein, a plant flavonoid, attacks the cell cycle at G2 and/or early M phases in most carcinoma cells. Quercetin and genistein block the phosphatidylinositol conversion to IP3 signal transduction pathway mainly by inhibiting 1-phosphatidylinositol 4-kinase (PI kinase, EC 2.7.1.67) and 1-phosphatidylinositol 4-phosphate 5-kinase (PIP kinase, EC 2.7.1.68), respectively. Because each drug attacks a different phase of the cell cycle and reduces IP3 concentration by attacking different signal transduction enzymes, we tested the hypothesis that the two drugs might be synergistic in human carcinoma cells. In human ovarian carcinoma OVCAR-5 cells in growth inhibition assay, the IC50S for quercetin and genistein were (mean +/- SE) 66 +/- 3.0 and 32 +/- 2.5 microM; in clonogenic assays they were 15 +/- 1.2 and 5 +/- 0.5 microM, respectively. When quercetin was added to the cultures of OVCAR-5 cells followed 8 h later by genistein, synergism was observed in growth inhibition and clonogenic assays. The synergistic action of quercetin and genistein may be of interest in clinical treatment of human ovarian carcinoma.     

Correct blue-light regulation of pea Lhcb genes in an Arabidopsis background

The phasic contraction of the isolated guinea pig vas deferens induced by adenosine 5'-triphosphate (ATP) 1mM was significantly augmented by acidification of bathing solution induced by administration of hydrochloric acid (HCL) 10mM (pH = 6.87 +/- 0.015 (n = 5); mean +/- S.E.), while the tonic contraction induced by norepinephrine (NE) 10microM was significantly depressed by HCl 10mM. The contractile response to ATP 1mM was markedly potentiated in the presence of NE 10 microM. The potentiated contractile response to ATP 1mM in the presence of NE 10microM was significantly augmented by HCl 1mM or 10mM. The potentiating ratio of the contraction induced by ATP 1mM in the presence of NE 10microM to that induced by ATP 1mM alone was almost unaffected by the administration of HCl. Electrical field stimulation (EFS) produced a biphasic contractile response of the isolated guinea pig vas deferens; viz. the first rapid phasic contractile response and the second slow maintained tonic contractile response. The phasic contractile response to EFS was significantly augmented by HCl 10mM. These results may indicate that acidification of the medium potentiates the neurochemical transmission between the nerve terminals and the smooth muscle of vas deferens via sensitization of P2X (presumably P2X1 or P2X2) receptors existing in the smooth muscle.     

A micromechanical model of lung tissue rheology

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Clinical spectrum of nonmenstrual toxic shock syndrome (TSS): comparison with menstrual TSS by multivariate discriminant analyses

1. The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4-dihydropyridine calcium antagonist. 2. We studied the effect of lacidipine on contraction evoked by 100 mM KCl solution in rat aorta, treated by N omega-nitro-L-arginine (0.1 mM), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)-isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine-evoked inhibition was a pseudo-first order association kinetics, we estimated the dissociation rate constant (k-1 = 0.031 min-1), the association rate constant (k1 = 2.70 x 10(8) M-1 min-1) and the dissociation constant (KD = k-1/k1 = 115 pM) which was close to the IC50 value in steady-state conditions (160 pM). 3. The inhibitory effects of lacidipine and (+)-isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mM KCl-solution. Washout with drug-free 40 mM K(+)-depolarizing solution reversed inhibition in the (+)-isradipine-treated preparations, but not in the lacidipine-treated ones. 4. Radioligand binding studies were performed with [3H]-lacidipine and [3H]-isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for[3H]-lacidipine: KD = 23 +/- 2.6 pM, Bmax = 380 +/- 21 fmol mg-1 protein in membranes from aorta; KD =23 +/- 3.1 pM, Bmax = 790 +/- 60 fmol mg-1 protein in membranes from ileum; for [3H]-isradipine:KD = 140 +/- 46 pM, Bmax = 350 +/- 64 fmol mg-1 protein in membrane from aorta; KD = 68 +/- 14 pM,Bmax = 760 +/- 75 fmol mg-1 protein in membranes from ileum). After isotopic dilution, [3H]-lacidipine and [3H]-isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (kappa_ 1) were 0.0257 min-1 and 0.0595 min-1, for [3H]-lacidipine and[3H]-isradipine, respectively.5. The non specific binding of [3H]-lacidipine and [3H]-isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine(1 microM). After incubation with [3H]-lacidipine 77.6 +/- 1.9 pM for 2 h the concentration of drug in the tissue was 15.15 +/- 1.18 fmol mg-1 w.wt. and still amounted to 7.24 +/- 0.61 fmol mg-1 w.wt. after 3.5 h washout in drug-free solution. After incubation with [3H]-isradipine 47.2 +/- 0.4 pM for 2 h it was 2.26 +/-0.07 fmol mg-1 w.wt. and was undetectable after 3.5 h washout in a drug-free solution.6. It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a nonspecific binding in a tissue compartment different from the plasma membrane.     

The effect of sodium based hypo-osmolality on arterial smooth muscle reactivity in vitro

The study tested the hypothesis that the reduced [Na+]e and hypo-osmolality of normal pregnancy are causally linked to the attenuation of vascular smooth muscle reactivity in vitro. Aortic rings from nonpregnant female rats were incubated in physiological medium containing 114 mM NaCl/l and the contractile responses to phenylephrine, KCl and CaCl2 as well as the relaxations to acetylcholine and KCl were compared with those of rings incubated in normal medium containing 119 mM NaCl/l. There was no solute substituted for the lowered [Na+]. Experiments with phenylephrine were repeated using de-endothelialized rings and intact rings pretreated with indomethacin. Contractile responses of intact rings (n = 11) in hypo-osmolar solution to phenylephrine were significantly (P < 0.001) lower than of those in normal medium (n = 11). Responses were partially restored by endothelial denudation but not in the presence of indomethacin. Relaxations to acetylcholine (n = 7 for hypo-osmolar; n = 6 for normal solution) and KCl (n = 7 for each of hypo- and normal osmolar) were significantly enhanced (P < 0.05) in rings incubated in hypo-osmolar solution. There was no significant difference between the responses of the rings to KCl, and CaCl2 in either solution. These effects are similar to some of those previously described for vascular smooth muscle in normal pregnancy suggesting that the reduced [Na+]e and hypo-osmolarity of normal pregnancy may be contributing to the diminished vascular reactivity.     

Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds

1. Ouabain, an inhibitor of Na+/K+ ATPase induces the release of acetylcholine from central and myenteric cholinergic neurones principally due to partial depolarization of the cell membrane. The effect of ouabain has been examined on neurogenic contractions in the guinea-pig ileum arising from either electrical field stimulation or from naloxone in morphine-exposed preparations. 2. Guinea-pig isolated ileum preparations were stimulated transmurally (0.1 Hz, 0.3 ms, 200 mA) to elicit contractions of the myenteric plexus-longitudinal smooth muscle. 3. Incubation with morphine (0.3 microM, 60 min) was followed by naloxone (1 microM) which produced withdrawal contractions in 16/26 preparations (median of 10.7 [2.2-40.0]% of a maximal contracture to KCl (60 mM)). 4. In parallel experiments, ouabain (1 microM) was added to the tissue before exposure to morphine (0.3 microM, 60 min). Naloxone (1 microM) subsequently displayed a withdrawal contraction in all 26/26 tissues (57.9 [30.5-151.7]% of a maximal contracture to KCl (60 mM). 5. Ouabain neither affected the concentration-dependent contractions of guinea-pig ileum produced by carbachol nor the inhibition of electrically-evoked contraction produced by morphine (0.3 microM). 6. The muscarinic antagonist atropine (0.1 microM) antagonized control naloxone withdrawal responses. The atropine resistant component, evident in ouabain-treated tissues, was blocked by SR140333((S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenyla cetyl)piperidin-3-yl]ethyl]-4-phenyl-1-azoniabicyclo[2.2. 2]-octane, chloride), a substance P antagonist. 7. Clonidine (alpha2-adrenoceptor agonist) inhibited electrically-evoked contractions. Exposure to the alpha2-adrenoceptor antagonist RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline), resulted in a contracture which was not significantly enhanced by ouabain (1 microM). 8. Ouabain selectively potentiates the naloxone-induced withdrawal contraction following acute exposure to morphine the major components of which are mediated by both acetylcholine and substance P.     

Effect of mycophenolic acid on TNF alpha-induced expression of cell adhesion molecules in human venous endothelial cells in vitro

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA). 2. In endothelium-intact strips, histamine (His, 3-10 microM) and high K+ (20-80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 microM), 5-hydroxytryptamine (10 microM) and 9,11-epithio-11, 12-methano-thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+-induced contraction). 3. N(G)-nitro-L-arginine (L-NOARG, 100 microM), but not indomethacin (10 microM), greatly enhanced the phasic and the tonic contractions induced by His (1-10 microM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3-3 microM) caused concentration-dependent relaxation (maximum relaxation by 89.7 +/- 7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 microM), but not glibenclamide (3 microM), abolished the relaxation induced by ACh (0.3-3 microM) in L-NOARG-treated strips (n=4, P<0.05). 4. In endothelium-intact tissues, His (3 microM) depolarized the smooth muscle membrane potential (by 4.4 +/- 1.8 mV, n = 12, P < 0.05) whereas ACh (3 microM) caused membrane hyperpolarization (-20.9 +/- 3.0 mV, n = 25, P< 0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (-23.5 +/- 5.9 mV, n=8, P<0.05) or glibenclamide (-20.6 +/- 5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to - 5.8 +/- 3.2 mV, n = 3, P< 0.05). 5. Sodium nitroprusside (0.1-10 microM) did not hyperpolarize the smooth muscle cell membrane potential (0.2 +/- 0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n-4, P<0.05). 6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.