Stimulation by menthol of Cl secretion via a Ca(2+)-dependent mechanism in canine airway epithelium.

1. To investigate the effect of menthol on airway epithelial ion transport function, we studied the bioelectrical properties of canine cultured tracheal epithelium by Ussing's short-circuit technique in vitro. 2. Addition of menthol (10(-3) M) to the mucosal but not the submucosal solution increased the short-circuit current (Isc) from 6.2 +/- 0.9 to 14.0 +/- 2.2 microA cm-2 (P < 0.001), and this effect was accompanied by increases in transepithelial potential difference and conductance. The response was dose-dependent, with the maximal increase from the baseline value and the concentration required to produce a half-maximal effect (EC50) being 6.4 +/- 0.9 microA cm-2 (P < 0.001) and 40 microM, respectively. 3. Other cyclic alcohols, including menthone and cyclohexanol, had no effect on the electrical properties. 4. The menthol-induced increase in Isc was not altered by pretreatment of the cells with amiloride, indomethacin, or propranolol but was abolished by diphenylamine-2-carboxylate, furosemide or substitution of Cl with iodide in the medium. 5. Menthol (10(-3) M) increased cytosolic levels of free calcium ([Ca2+]i) from 98 +/- 12 to 340 +/- 49 nM (P < 0.01) in fura-2-loaded tracheal epithelium but did not affect the intracellular adenosine 3',5'-cyclic monophosphate content. 6. These results suggest that menthol stimulates Cl secretion across airway epithelium, probably through a Ca(2+)-dependent mechanism, and might thus influence mucociliary transport in the respiratory tract.     

Adenosine induces cyclic-AMP formation and inhibits endothelin-1 production/secretion in guinea-pig tracheal epithelial cells through A(2B) adenosine receptors

1. The adenosine receptor subtype mediating adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation and the effect of its activation on endothelin-1 (ET-1) secretion were studied in primary cultures of tracheal epithelial cells. 2. Adenosine analogues showed the following rank order of potency (pD(2) value) and intrinsic activity on the generation of cyclic AMP by tracheal epithelial cells: 5'-N-ethylcarboxyamidoadenosine (NECA, A(1)/A(2A)/A(2B), pD(2): 5.44+/-0.16)>adenosine (ADO, non selective, pD(2): 4.99+/-0. 09; 71+/-9% of NECA response) >/=2-Cl-adenosine (2CADO, non selective, pD(2): 4.72+/-0.14; 65+/-9% of NECA response)>CGS21680 (A(2A); inactive at up to 100 microM). 3. Cyclic AMP formation stimulated by NECA in guinea-pig tracheal epithelial cells was inhibited by adenosine receptor antagonist with the following order of apparent affinity (pA(2) value): Xanthine amine congeners (XAC, A(2A)/A(2B), 7.89+/-0.22)>CGS15943 (A(2A)/A(2B), 7.24+/-0. 26)>ZM241385 (A(2A), 6.69+/-0.14)>DPCPX (A(1), 6.51+/-0. 14)>3n-propylxanthine (weak A(2B), 4.30+/-0.10). This rank order of potency is typical for A(2B)-adenosine receptor. 4. Adenosine decreased basal and LPS-stimulated irET production in a concentration-dependent manner. Moreover, NECA but not CGS21680 inhibited LPS-induced irET production. 5. The inhibitory effect of NECA on LPS-induced irET production was reversed by XAC (pA(2)=8.84+/-0. 12) and DPCPX (pA(2)=8.10+/-0.22). 6. These results suggested that adenosine increased cyclic AMP formation and inhibited irET production/secretion by guinea-pig tracheal epithelial cells through the activation of a functional adenosine receptor that is most likely the A(2B) subtype. This adenosine receptor may be involved in the regulation of the level of ET-1 production/secretion by guinea-pig tracheal epithelial cells in physiological as well as in pathophysiological conditions.     

A possible role for cyclic adenosine 3'',5''-monophosphate in the regulation of acid secretion in the isolated stomach of guinea-pig.

1 The rate of acid secretion and mucosal cyclic adenosine 3',5'-monophosphate (cyclic AMP) content have been measured on the same guinea-pig isolated stomach preparation in response to histamine, theophylline and ICI 63197, a potent phosphodiesterase inhibitor. 2 Unstimulated control tissues had a spontaneous rate of acid secretion of 74.41 +/- 9.06 mumol H+/g wet wt. of mucosa per hour (s.e. mean, n = 20) and a cyclic AMP content of 0.517 +/- 0.058 mnol/g wet weight. 3 Each of the three drugs caused an increase in both the mucosal cyclic AMP content and the rate of acid secretion. These increases were inearly related to the logarithm of drug concentration for each drug. 4 There were no statistically significant differences between the three regression coefficients obtained for acid on drug and for cyclic AMP on drug. 5 There was a significant correlation between the rate of acid secretion and mucosal cyclic AMP content in stimulated preparations (P less than 0.001) and also in control preparations which received no drug (P less than 0.05). 6 These results are discussed in relation to the possible role of cyclic AMP in the mediation of acid secretory responses in the mammalian stomach.     

Influence of the epithelium on responsiveness of guinea-pig isolated trachea to contractile and relaxant agonists.

The potency (pD2) and maximal contractile effect (Emax) of histamine, acetylcholine, carbachol and K+ were assessed from cumulative concentration-effect curves in guinea-pig isolated tracheal ring preparations with and without an intact epithelium. Estimates of Emax were not significantly different in epithelium-denuded preparations compared with those measured in intact preparations; pD2 values for acetylcholine, carbachol and K+ were not significantly altered. In contrast, the potency of histamine was significantly increased by about 4 fold in preparations devoid of epithelial cells. Estimates of potency and Emax were also determined for the smooth muscle relaxants isoprenaline, forskolin and theophylline (which increase intracellular cyclic AMP) and for nitroglycerin (which increases cyclic GMP) in both intact and epithelium-stripped tracheal rings. The pD2 values for these relaxants were not significantly altered by the removal of the epithelium. However, with the exception of nitroglycerin, Emax values for these relaxants were significantly lower in stripped than in intact tracheal rings that had been maximally precontracted with carbachol. The autoradiographic localisation of binding sites for the non-selective beta-adrenoceptor ligand [125I]-iodocyanopindolol (I-CYP) showed that the epithelium of the guinea-pig trachea had a 75 +/- 16% greater density of beta-adrenoceptors than the smooth muscle. Removing the epithelium did not significantly alter either the density of smooth muscle binding sites or the affinity of I-CYP binding. It was concluded that the reduced functional response of guinea-pig trachea to isoprenaline was probably not due to smooth muscle beta-adrenoceptor dysfunction. Results indicate that the epithelium plays an important role in the modulation of responsiveness of guinea-pig trachea to histamine and relaxants that mediate their effects by selectively increasing intracellular cyclic AMP levels.     

Relaxant effect of pituitary adenylate cyclase-activating polypeptide on guinea-pig tracheal smooth muscle.

We investigated the relaxant effect of the pituitary adenylate cyclase-activating polypeptide with 27 residues (PACAP27) and with 38 residues (PACAP38) on guinea-pig tracheal smooth muscle. Both forms of PACAP showed dose-dependent relaxant effects. The EC50 of PACAP27 was 8.7 +/- 1.9 x 10(-8) M and that of PACAP38 was 6.8 +/- 1.0 x 10(-8) M. Both increased cyclic AMP levels dose dependently and the elevation of cyclic AMP preceded the relaxation of tracheal smooth muscle. There was a marked difference in the duration of action of the two peptides. PACAP38 showed a longer-lasting relaxation compared to PACAP27. Furthermore PACAP38 maintained significantly higher levels of cyclic AMP, with cyclic AMP levels at 60 min after a 5-min exposure to PACAPs (10(-6) M) being 14.0 +/- 1.4 pM/mg protein for PACAP27 and 35.9 +/- 2.4 pM/mg protein for PACAP38. These results suggest that PACAP27 and PACAP38 may be novel potent relaxants in tracheal smooth muscle and their relaxant effect might be mediated by cyclic AMP. However PACAP38 had a longer-lasting action on relaxation of tracheal smooth muscle and production of tissue cyclic AMP than PACAP27.     

Influence of vasoactive intestinal polypeptide on net water flux and cyclic adenosine 3′,5′-monophosphate formation in the rat jejunum

1. The effects were studied of vasoactive intestinal polypeptide (VIP), theophylline, and morphine on net water flux and cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels in the jejunum of anaesthetized rats in vivo and of VIP and morphine on adenylate cyclase activity in rat epithelial cell membranes in vitro. 2. Infusion of VIP (0.1-2 x 10(-9) mol/min/kg) dose dependently caused a reversal from net water absorption to net secretion; 2 x 10(-9) mol/min/kg enhanced the mucosal cyclic AMP content by 67%. 3. Theophylline (5 mg/ml, intraluminally) enhanced the effect of intra-arterial infusion of VIP (2 x 10(-9) mol/min/kg) as to net water secretion and increase in mucosal cyclic AMP content. 4. Pretreatment with morphine (5 mg/kg, s.c.) did not influence the effects of VIP on net water flux and on mucosal cyclic AMP content. 5. Atropine (2 mg/kg, s.c.) also failed to reduced the effect of VIP (0.4 x 10(-9) mol/min/kg) on net water flux. 6. Stimulation of adenylate cyclase activity was a function of VIP concentration over a range of 1 x 10(-10)-1 x 10(-7) M. Morphine (up to 1 x 10(-3) M) failed to influence stimulation of adenylate cyclase by VIP. 7. The finding that low doses of VIP, which already have an effect on net water flux, fail to increase cyclic AMP levels makes it likely that other mediators besides cyclic AMP are involved in the effect of VIP on net water flux. Some of the present results, however, support the assumption that VIP stimulates intestinal fluid secretion by increasing mucosal cyclic AMP levels.     

Possible role of cyclic AMP phosphodiesterases in the actions of ibudilast on eosinophil thromboxane generation and airways smooth muscle tone.

1. The possible role of cyclic AMP phosphodiesterase (PDE) in the inhibitory actions of ibudilast on tracheal smooth muscle contractility and eosinophil thromboxane generation was investigated. 2. Ibudilast was a non-selective inhibitor of partially purified cyclic nucleotide PDE isoenzymes from pig aorta and bovine tracheal smooth muscle, exhibiting only moderate potency against bovine tracheal PDE IV (IC50 = 12 +/- 4 microM, n = 3). Similar or slightly lower potencies were displayed against PDEs I, II, III and V. In contrast, rolipram exhibited selectivity for PDE IV (3 +/- 0.5 microM, n = 3). 3. Ibudilast (IC50 = 0.87 +/- 0.37 microM, n = 3), like rolipram (IC50 = 0.20 +/- 0.04 microM, n = 3), was a more potent inhibitor of membrane-bound PDE IV from guinea-pig eosinophils than of partially purified PDE IV from bovine tracheal smooth muscle. The potency of ibudilast increased when the eosinophil enzyme was solubilised with deoxycholate and NaCl (IC50 = 0.11 +/- 0.05 microM, n = 3) or exposed to vanadate/glutathione complex (V/GSH) (IC50 = 0.11 +/- 0.02 microM, n = 3). The potency of rolipram was also increased by solubilization (IC50 = 0.012 +/- 0.003, n = 3) or V/GSH (IC50 = 0.012 +/- 0.003, n = 3). 4. In intact eosinophils, ibudilast (0.032 microM-20 microM) potentiated isoprenaline-induced cyclic AMP accumulation in a concentration-dependent manner, being approximately 20 fold less potent than rolipram. Little or no effect on basal cyclic AMP levels was observed with either compound.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of histamine on cyclic AMP levels in guinea-pig tracheal smooth muscle

Histamine (10(-5)--3 x 10(-4) M) increased the cylic AMP content of guinea-pig tracheal smooth muscle, the maximal effect being a 3-fold increase after 2-min incubation with 10(-4) histamine. Histamine-induced accumulation of cyclic AMP was not affected by propranolol or atropine, but was reduced by mepyramine. Aspirin and indomethacin abolished the cyclic AMP response to histamine and potentiated histamine-induced contractions of the smooth muscle. These results suggest that the elevation of cyclic AMP levels in response to histamine is mediated by prostaglandins, and represents an important negative feedback regulatory mechanism which modulates the contractile response of guinea-pig tracheal smooth muscle to histamine.     

Functional interaction of a beta-adrenergic agonist and cyclic GMP phosphodiesterase inhibitor in control and hypertrophic cardiomyocytes

This study tested the hypothesis that the positive inotropic effect of beta-adrenoceptor stimulation would be inhibited by increases in cyclic GMP in control cardiomyocytes and that this response would be modified in hypertrophic cardiomyocytes. Cell functional data as well as GMP and cyclic AMP data were collected from 7 control and 7 1K1C (one-kidney-one-clip) renal hypertensive hypertrophic rabbits. Using isolated control and IKIC ventricular myocytes, data were obtained at baseline and after treatment with the beta-adrenoceptor agonist isoproterenol (10(-8, -6) mol/l) or the cyclic GMP-phosphodiesterase inhibitor zaprinast (10(-5) mol/l) followed by isoproterenol (10(-8, -6) mol/l). We found that in control rabbits, isoproterenol (10(-6) mol/l) increased percent shortening (4.8 +/- 0.2 to 6.4 +/- 0.3%) and cyclic AMP (2.3 +/- 0.3 to 5.0 +/- 0.7 pmol/10(5) cells). Zaprinast 10(-5) mol/l increased cyclic GMP (150 +/- 20 to 209 +/- 14 fmol/10(5) cells) and decreased percent shortening (6.2 +/- 0.4 to 5.2 +/- 0.3). Zaprinast 10(-5) mol/l prevented the functional response to isoproterenol in control (5.2 +/- 0.3 to 4.7 +/- 0.3), without changing cyclic AMP levels. In 1K1C rabbits, isoproterenol (10(-6) mol/l) increased cyclic AMP (4.9 +/- 0.8 to 7.6 +/- 1.4 pmol/10(5) cells) without changing function. Zaprinast 10(-5) mol/l increased cyclic GMP (182 +/- 23 to 233 +/- 24 fmol/10(5) cells) and decreased percent shortening (6.6 +/- 0.9 to 4.7 +/- 0.5), but did not alter the lack of effect of isoproterenol in 1K1C. In control cardiomyocytes, cyclic GMP blunted the isoproterenol contraction response without changing cyclic AMP levels, but isoproterenol's functional effect was not seen in 1K1C cardiomyocytes.     

Depressive effect of epinephrine mediated via adrenergic beta-receptor in isolated rat colon and duodenum.

Differences in sensitivity to catecholamines between colon and duodenum were examined in tissues from the rat, monitoring the depressive effect of catecholamines on contractile response to acetylcholine (ACh). The sensitivity of colonic tissue to ACh was higher than that of duodenal. Epinephrine (Ep, 10(-7) g/ml) depressed the contractile response to ACh in the colonic tissue, but not in the duodenal. The depressive effect of Ep on the contractile response to ACh is attributed to the stimulation of adrenergic beta-receptors in the colonic tissue as the depression disappeared by pretreatment with propranolol (10(-6) g/ml). There was no difference on the depressive effect of papaverine on the contractile response to ACh, except when low concentrations were used. Dibutyryl cyclic AMP (10(-4) g/ml) depressed the contractile responses of both tissues to ACh. After treatment with Ep (10(-7) g/ml), cyclic AMP content was increased in the colonic tissue but not in the duodenal. However, papaverine (3 X 10(-6 g/ml) and a higher dose of Ep(10(-6) g/ml) increased cyclic AMP content in both tissues. The increase of cyclic AMP and the decrease of tension caused by Ep were not correlated in these tissues. However, a positive correlation was observed between the depressive effect of Ep on the contractile response to ACh and the increase of cyclic AMP content in these tissues.     

Role of the epithelium in the control of intestinal motility: implications for intestinal damage after anoxia and reoxygenation.

A vibration technique was used to dislocate the epithelium from the rat small intestine, in order to study the possible regulatory role of the epithelium on intestinal motility. Complete removal of the epithelium led to a slightly potentiated contraction of the longitudinal smooth muscle by the muscarinic agonist methacholine (pD2. 6.5 +/- 0.1 vs. 6.2 +/- 0.2). The maximal beta-adrenergic response expressed relative to the relaxation by 0.5 mM dibutyryl cyclic AMP increased from 55.9 +/- 9.0% to 72.6 +/- 9.1% by this treatment. Efforts were made to relate these observations to the endothelium-dependent relaxation in blood vessels, but no indication was found for a similar mechanism in the small intestine. Not only mechanical dislocation can be employed to affect the mucosal layer, but also intestinal ischemia has been reported to lead to mucosal damage. In this study we mimicked ischemia by applying in vitro anoxia and subsequent reoxygenation to isolated intestinal segments. When intestinal segments are isolated and kept in physiological buffer, xanthine dehydrogenase is converted slowly to xanthine oxidase, irrespective of whether the buffer is oxygenated or not. No evidence was found for oxygen radical damage after anoxia and reoxygenation. However, the intestinal mucosa was damaged both after normoxia, and after anoxia and reoxygenation. Anoxia and subsequent reoxygenation did not affect muscarinic contraction, but slightly increased the beta-adrenergic relaxation, which partly correlates with the effects of mechanical dislocation of the epithelium. The increased sensitivity of the smooth muscle after epithelial damage might be involved in motility changes during intestinal inflammatory diseases.     

The identification of apparently novel cyclic AMP and cyclic GMP phosphodiesterase activities in guinea-pig tracheal smooth muscle.

Phosphodiesterase (PDE) activities that were capable of hydrolysing cyclic AMP (Km = 6.8 +/- 2 microM) and cyclic GMP (Km = 6.7 +/- 1.6 microM) were isolated from tracheal smooth muscle. These enzyme(s) activities were insensitive to stimulation by calcium/calmodulin and to inhibition by cyclic GMP, rolipram (type IV inhibitor) and siguazodan (type III inhibitor). Zaprinast was a relatively poor inhibitor of both cyclic AMP and cyclic GMP hydrolysis (IC50 = 46 +/- 9 microM and 45 +/- 14 microM respectively). These results suggest that tracheal smooth muscle may contain an apparently novel PDE. However, KCl (30 mM) which facilitates calcium entry in cells, depressed bradykinin-stimulated intracellular cyclic AMP formation, suggesting that the type I PDE may be functionally present. We suggest that considerable caution be exercised in identifying apparently novel PDE isoforms.     

Effect of 1,4-dimorpholino-7-(4-hydroxyphenyl)pyrido[3,4-d]) pyridazine [DS-511(4'-OH)] on the transepithelial transport of sodium and water and the permeability to urea in the toad urinary bladder.

To elucidate the mechanism of the inhibitory action of 1,4-dimorpholino-7-phenylpyrido[3,4-d]pyridazine (DS-511) on water and sodium reabsorption at the renal tubules, the effect of DS-511 (4'-OH), which is similar in diuretic effect to but more water-soluble than DS-511, on the transepithelial transport of sodium and water and permeability to urea was studied in isolated toad urinary bladder. Application of DS-511(4'-OH) at concentrations above 2 x 10(-4) mol/l to the serosal side of the bladder depressed the transepithelial potential difference, short circuit current (SCC), and membrane conductance as well as the increased response of the SCC to arginine vasopressin (AVP) and cyclic AMP. The effect of DS-511 (4'-OH) applied to the mucosal side was delayed in onset and less pronounced. Neither serosal nor mucosal 10(-3) mol/l DS-511(4'-OH) depressed the increased response of the SCC to amphotericin B. 2 x 10(-4) mol/l DS-511 (4'-OH) applied to the serosal side did not affect osmotic water flow, but potentiated the increase in water flow caused by AVP. Basal urea permeability as well as the increase in urea permeability caused by AVP were depressed by serosal 10(-3) mol/l DS-511 (4'-OH). The results show that DS-511(4'-OH) has two actions, the depression of the transepithelial transport of sodium and urea, and the potentiation of the increased water permeability caused by AVP.     

Subsensitivity to cholinoceptor stimulation of the human iris sphincter in situ following acute and chronic administration of cholinomimetic miotic drugs.

1 The effects were studied of prostaglandin E1 (PGE1), theophylline and morphine on net water flux and mucosal cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels in the jejunum of anaesthetized rats in vivo. 2 Infusion of PGE1 (3.2 micrograms/min, i.a.) caused a reversal from net water absorption to net secretion and enhanced the mucosal cyclic AMP content by 54%. 3 Theophylline (5 mg/ml, intraluminal) similarly produced a reversal from net water absorption to net secretion and increased mucosal cyclic AMP content by 54%. Additional intra-arterial infusion of PGE1 resulted in a massive increase in net water secretion and an increase in mucosal cyclic AMP content by about 200%. 4 Pretreatment with morphine (10 mg/kg, s.c.) reduced the effect of PGE1 on net water flux and completely inhibited its effect on the mucosal cyclic AMP content. Naloxone (10 mg/kg, s.c.) abolished both effects of morphine. 5 A good correlation (r = 0.99) was demonstrated between mucosal cyclic AMP levels and net water flux. 6 The present results demonstrate that PGE1 stimulates intestinal fluid secretion by increasing mucosal cyclic AMP levels. The antidiarrhoeal effect of morphine can be explained by its inhibition of the PGE-mediated increase in cyclic AMP levels, which, in turn, leads to a reduction in intestinal secretion.     

Prostaglandin E(2) increases cyclic AMP and inhibits endothelin-1 production/secretion by guinea-pig tracheal epithelial cells through EP(4) receptors

Prostaglandin E(2) (PGE(2)) increased adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation in tracheal epithelial cells and concomitantly decreased the production/secretion of immunoreactive endothelin (irET). Naturally occurring prostanoids and selective and non-selective EP receptor agonists showed the following rank order of potency in stimulating cyclic AMP generation by epithelial cells: PGE(2) (EP-selective)>16,16-dimethyl PGE(2) (EP-selective)>11-deoxy PGE(2) (EP-selective)>iloprost (IP/EP(1)/EP(3)-selective), butaprost (EP(2)-selective), PGD(2) (DP-selective), PGF(2alpha) (FP-selective). The lack of responsiveness of the latter prostanoids indicated that the prostanoid receptor present in these cells is not of the DP, FP, IP, EP(1), EP(2) or EP(3) subtype. Pre-incubating the cells with the selective TP/EP(4)-receptor antagonists AH23848B and AH22921X antagonized the PGE(2)-evoked cyclic AMP generation. This suggested that EP(4) receptors mediate PGE(2) effects. However, in addition to any antagonistic effects at EP(4)-receptors, both compounds, to a different extent, modified cyclic AMP metabolism. The selective EP(1), DP and EP(2) receptor antagonist (AH6809) failed to inhibit PGE(2)-evoked cyclic AMP generation which confirmed that the EP(2) receptor subtype did not contribute to the change in cyclic AMP formation in these cells. The PGE(2)-induced inhibition of irET production by guinea-pig tracheal epithelial cells was due to cyclic AMP generation and activation of the cyclic AMP-dependent protein kinase since this effect was reverted by the cyclic AMP antagonist Rp-cAMPS. These results provide the first evidence supporting the existence of a functional prostaglandin E(2) receptor that shares the pharmacological features of the EP(4)-receptor subtype in guinea-pig tracheal epithelial cells. These receptors modulate cyclic AMP formation as well as ET-1 production/secretion in these cells.     

Endothelin stimulates short circuit current in a cultured epithelium.

1. Endothelin, a novel potent vasoconstrictor peptide produced by vascular endothelial cells stimulated anion secretion by a cultured secretory epithelium derived from the rat epididymis as measured by changes in short-circuit current (SCC). 2. Stimulation of the SCC was observed when endothelin was added to the basolateral or the apical side of the epithelium. The response to basolateral application was greater than that to apical application. The EC50 values were found to be 1.3 nM and 3.0 nM for basolateral and apical application, respectively. These values were about half an order to one order of magnitude higher than that required for its vasoconstrictor action. 3. The stimulation of SCC by endothelin was accompanied by a rise in the intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) content in epididymal monolayers. 4. The effect of endothelin on SCC was mediated through an increase in prostaglandin synthesis by the tissues and was not blocked by an antagonist of angiotensin II (Sar1 Ile8 angiotensin II) or of adrenaline (propranolol). 5. It is speculated that endogenous endothelin plays an important role in the control of water and electrolyte transport in the epididymis.     

Activation of nitric oxide synthase by beta 2-adrenoceptors in human umbilical vein endothelium in vitro.

1. Some animal studies suggest that beta-adrenoceptor-mediated vasorelaxation is in part mediated through nitric oxide (NO) release. Furthermore, in humans, we have recently shown that forearm blood flow is increased by infusion of beta2-adrenergic agonists into the brachial artery, and the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) inhibits this response. 2. The purpose of the present study was to determine whether stimulation of human umbilical vein endothelial beta-adrenoceptors causes vasorelaxation and nitric oxide generation, and whether this might be mediated by cyclic adenosine-3',5'-monophosphate (cyclic AMP). 3. Vasorelaxant responses were determined in umbilical vein rings to the nonselective beta-adrenergic agonist isoprenaline and to the cyclic AMP analogue dibutyryl cyclic AMP, following precontraction with prostaglandin F2alpha. 4. NOS activity was measured in cultured human umbilical vein endothelial cells (HUVEC) by the conversion of [3H]-L-arginine to [3H]-L-citrulline, and adenylyl cyclase activity by the conversion of [alpha-32P]-ATP to [32P]-cyclic AMP. 5. Isoprenaline relaxed umbilical vein rings, and this vasorelaxation was abolished by beta2- (but not beta1-) adrenergic blockage, and by endothelium removal or 1 mM L-NMMA. In addition, vasorelaxant responses to dibutyryl cyclic AMP were inhibited by 1 mM L-NMMA, with a reduction in Emax from 90.0+/-9.3% to 50.5+/-9.9% (P<0.05). 6. Isoprenaline 1 microM increased NOS activity in HUVEC (34.0+/-5.9% above basal, P<0.001). Furthermore, isoprenaline increased adenylyl cyclase activity in a concentration-dependent manner; this response was inhibited by beta2 (but not beta1-) adrenergic blockade. Forskolin 1 microM and dibutyryl cyclic AMP 1 mM each increased NOS activity in HUVEC, to a degree similar to isoprenaline 1 microM. The increase in L-arginine to L-citrulline conversion observed with each agent was abolished by coincubation with NOS inhibitors. 7. These results indicate that endothelial beta2-adrenergic stimulation and cyclic AMP elevation activate the L-arginine/NO system, and give rise to vasorelaxation, in human umbilical vein.     

Calcium- and cyclic AMP-dependent chloride secretion in human colonic epithelia

Three stable epithelial cell lines (HCA-7, HCA-7-Col 1 and HCA-7-Col 3) all derived from the same human adenocarcinoma have been cultured on collagen-coated Millipore filters. These epithelial monolayers have been used to record short circuit current (SCC) in response to of secretagogues. Similar monolayers, but grown on plastic dishes, were used for measurements of tissue cyclic AMP. Lysylbradykinin, applied to either side of the monolayers, increased SCC in HCA-7 cells but had little effect on the other two lines. The responses showed rapid desensitization, which could be prevented by cooling to 4 degrees C. Responses to kinin were not significantly attenuated by piroxicam, an inhibitor of cyclo-oxygenase. Other secretagogues, vasoactive intestinal polypeptide (VIP) and carbachol also increased SCC in monolayers. The responses to VIP were greatest in HCA-7-Col 1 monolayers while responses were virtually absent in HCA-7-Col 3. A similar profile was seen with carbachol except that responses of HCA-7 and HCA-7-Col 1 monolayers were more equal. With one exception the responses to VIP and carbachol showed sidedness, acting only from the basolateral side. The effects of the secretagogues were inhibited by piretanide, a loop diuretic, applied basolaterally. It is presumed that SCC responses represent electrogenic chloride secretion. Treatment with forskolin increased SCC in HCA-7 and HCA-7-Col 1 monolayers with little effect in HCA-7-Col 3. Nevertheless cyclic AMP levels were elevated most in HCA-7-Col 3 and least in HCA-7-Col 1 monolayers, in reciprocal relationship to the functional response. A23187 increased SCC when applied to HCA-7 and HCA-7-Col 3 monolayers with little effect on HCA-7-Col 1. The differential responses of the three human cell lines provide unique opportunities to discover the functional responsibilities of entities involved in the chloride secretory process. HCA-7-Col 3 cells which generate high levels of cyclic AMP in response to forskolin but which fail to show a substantial chloride secretory response may be a useful model of some disease conditions.     

Japanese herbal medicine Toki-shakuyaku-san (TJ-23) enhances cardiac contractile function in isolated ventricular cardiomyocytes

Toki-shakuyaku-san (TJ-23), a Japanese traditional herbal medicine, has a long history in Asia for the treatment of neurodegenerative, immune, and airway diseases. However, the effect of TJ-23 on heart function has not been elucidated. This study was designed to examine the effect of TJ-23 on ventricular contractile function at the single cardiomyocyte level. Ventricular cardiomyocytes from adult rat hearts were stimulated to contract at 0.5 Hz, and mechanical properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), and maximal velocity of shortening/relengthening (+/-dL/dt). TJ-23 (10(-)(8) - 10(-)(5) mg/ml) exhibited significant augmentation in PS, with a maximal response of 27.2%. TJ-23 at 10(-)(7) - 10(-)(5) mg/ml also increased +/-dL/dt, shortened TR(90), while had no effect on TPS. Pretreatment with the Na(+)-K(+)-ATPase inhibitor ouabain (1 microM), removal of extracellular sodium from contractile buffer (which inhibits Na(+)/Ca(2+) exchanger), or both concurrently abolished the positive effect of TJ-23 in cell shortening without inhibiting the baseline cell shortening. This study demonstrated a direct cardiac stimulatory action of TJ-23 at the cardiomyocyte level, which may be related to, at least in part, a Na(+)/K(+)-ATPase and/or Na(+)/Ca(2+) exchanger-dependent mechanism.     

Modulation of chemokine expression on intestinal epithelial cells by Kampo (traditional Japanese herbal) medicine, Hochuekkito, and its active ingredients

The intestinal epithelial cells sit at the interface between a lumen and a lamina propria or lymph nodes such as Peyer’s patches, where they play important roles in maintaining intestinal homeostasis through chemokine secretion. This study investigated the effect of Hochuekkito (TJ-41)—a traditional Japanese herbal (Kampo) formula used as a tonic for weakness—on chemokine expression in intestinal epithelial cells in order to explore the mechanism of its modulating effect against mucosal immunity. When cells from the rat normal small intestinal epithelial cell-line IEC-6 were stimulated with TJ-41, mRNA expression of CC chemokine ligand (CCL) 11 (eotaxin), CCL20 (MIP-3α) and CCL25 (TECK) was enhanced. Oral administration of TJ-41 to methotrexate-treated mice enhanced mRNA expression of CCL25 and keratinocyte growth factor in the jejunum with, decreasing mRNA expression of the inflammatory marker tumor necrosis factor (TNF)-α. Although oral administration of TJ-41 did not affect CCL20 mRNA expression in villus epithelium of methotrexate-treated mice, enhancement of CCL20 mRNA expression was observed in Peyer’s patches. Immunohistochemical analysis detected dense staining with anti-CCL20 antibody in the follicle-associated epithelium region of Peyer’s patches in mice administered TJ-41. Analysis of active ingredients indicates that polysaccharide-containing macromolecules in TJ-41 contribute to the enhancement of CCL20 mRNA expression through an intracellular signal cascade via nuclear factor kappa B (NF-κB) activation.