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The effects of one of the main components of fish oil, docosahexaenoic acid (DHA), on prostaglandin (PG) and Ca2+ signaling pathways were examined in intact mucosa and freshly isolated crypt cells of rabbit descending colon. Preincubation of serosal mucosa for 20 min with 1 microM DHA fully suppressed the short-circuit and transepithelial conductance increase induced by serosal addition of 10 microM arachidonic acid (AA). DHA at 1 microM also prevented the Cl- secretion promoted by 10 microM AA, as estimated by unidirectional 36Cl flux measurements (net flux = 0.68 +/- 0.30 versus -1.91 +/- 0.20 microEq/hr/cm2, four experiments, p0.001), whereas it did not affect the electrophysiological and ion flux responses to PGE2. Addition of 1 microM DHA to the serosal side of the mucosa also inhibited the PG cascade activation elicited by AA (PG synthesis and second messenger cAMP increase). In vitro assays of colonic cyclooxygenase activity showed that 1 microM DHA inhibited (with a 20-min lag) cyclooxygenase activity to the same extent as 5 microM indomethacin (approximately 82% and 80%, respectively). DHA also affected the Ca2+ signaling pathway; in isolated crypt cells, the cytosolic free Ca2+ concentration ([Ca2+]i) dropped by 49 +/- 7.6% (mean +/- standard error, six experiments) after incubation with 1 microM DHA. The sustained phase of the [Ca2+]i response to 500 nM concentrations of the intracellular Ca(2+)-ATPase inhibitor thapsigargin was also inhibited within 150 sec upon 1 microM DHA addition (141 +/- 5.8 versus 243 +/- 8.2 nM [Ca2+]i mean +/- standard error, eight experiments, p0.01). The [Ca2+]i-lowering effect of DHA, which was not achieved by incubation with other free fatty acids, was not prevented by removal of Na+ from the incubation medium (-46 +/- 4.3% versus -47 +/- 3.8%, mean +/- standard error, four experiments), nor it was mediated by cAMP-, protein kinase C-, or calmodulin-dependent mechanisms. The incubation of highly purified basolateral membranes of crypt cells with 1 microM DHA for 1 min produced a 5-fold increase (IC50 = 0.25 microM) in the plasma membrane Ca(2+)-ATPase activity (34.3 +/- 2.73 versus 6.02 +/- 0.50 nmol/mg of protein/min, mean +/- standard error, four experiments, p0.0001), thus indicating that the DHA effects on the Ca2+ pathway were mediated mainly by an increase in plasma membrane Ca2+ pump activity. These findings suggest that DHA is a powerful modulator of the cellular response to activation of PG and Ca2+ signaling pathways.

Summary: K + channel activators represent a novel class of smooth muscle relaxant agents. There is now much evidence demonstrating that K + channels, localized to prejunctional neurones and post-junctional smooth muscle membranes, can regulate airway smooth muscle activity, inducing smooth muscle cell membrane hyperpolarization. K + channel activity may be influenced by some neurotransmitters, such as adenosine, serotonin and noradrenaline. More recently, it has been observed that the stimulation of GABA B receptors influences K + channels in the hippocampus, dorsal rafe and spinal cord neurons. The aim of this study was to investigate the effects of levcromakalim in guinea-pig trachea at pre- and post-junctional sites and to evaluate whether GABA B receptors may modulate K + channel activation. Levcromakalim (from 1 nM to 1 μM) relaxed guinea-pig trachea (IC 50 10±0.9 nM) previously contracted by KCl (30 mM). This effect was reversed by a pretreatment with tetraethylammonium (10 mM) (IC 50 120±0.7 nM). A 30-min pretreatment with baclofen (1 μM) or phaclofen (1 μM) failed to modify the effects of levcromakalim (IC 50 18±1.0 nM and 14±0.6 nM, respectively). The contractile responses to electrical field stimulation (71.20±5.12% of acetylcholine -100 μM-contraction) was significantly ( P P P + channels may be localized both to prejunctional and post-junctional post-ganglionic sites, and may play an important role in modulating airway responsiveness. In fact, they may act at the prejunctional neurones, inhibiting the release of contractile neurotransmitter, or at the post-junctional sites, hyperpolarizing smooth muscle airway membranes. Moreover, the present study seems to demonstrate that K + channels localized on the prejunctional post-ganglionic nerves may be influenced by GABA B receptors.

The goal of the present study was to identify sites in the medulla oblongata where metabotropic glutamate receptors are involved in regulating respiration. Unilateral microinjections (50 nl) of l -glutamate ( l -glu) (10–25–50 mM) into the nucleus tractus solitarii (NTS) of anaesthetized rats elicited apnea (8.6 ± 0.3 sec; 21.3 ± 3.6 sec; 66.3 ± 16.5 sec respectively; N = 6) and arterial hypotension (7.3 ± 2.4 mmHg; 10.1 ± 2.3 mmHg; 35.3 ± 7.5 mmHg respectively; N = 6). Similarly, in other rats 1-aminocyclopentane-1, 3-dicarboxylic acid (ACPD) (1–5–10 mM), a selective agonist of metabotrophic glutamate receptors, also induced apnea (22.4 ± 2.5 sec; 32.5 ± sec; 92.5 ± 1.4 sec respectively; N = 6) and arterial hypotension (12.7 ± 2.2 mmHg; 19.6 ± 4.3 mmHg; 26.5 ± 1.5 mmHg respectively; N = 6). Paired experiments showed that unilateral microinjections of l -glu (50 mM) and ACPD (1 mM) into the nucleus retroambigualis (NRA) of anaesthetized rats elicited apnea (20.2 ± 2.6 sec and 33.8 ± 3.2 sec respectively; N = 6) and arterial hypotension (15.7 ± 3.7 mmHg and 22.5 ± 4.5 mmHg respectively; N = 6). The ACPD effects on apnea and hypotension in NTS and NRA were not prevented by a 3 min pretreatment with L-AP3 (30 mM), a putative antagonist of metabotropic glutamate receptors (19.5 ± 1.4 sec; 12.3 ± 3.2 mmHg and 30.6 ± 2.9 sec; 23.4 ± 3.8 mmHg respectively; N = 6). These data suggest that metabotropic glutamate receptors are involved in NTS and NRA regulation of cardiorespiratory functions. Moreover, these findings may also indicate that at particular doses L-AP3 could be considered a partial agonist of the metabotropic glutamate receptors in the medulla oblongata in anaesthetized rats.

The initial rate of [1-14C]arachidonic acid (AA) entry in the serosal side of rabbit distal colonic mucosa mounted in Ussing-type chambers is linear and independent of intracellular metabolism. When the maximal AA uptake was plotted as a function of medium AA concentration in ranges between 50 and 500 nM, saturation of the AA uptake with increasing concentrations was observed. The time course of the uptake of oleic acid and palmitic acid was similar to that observed with AA, and their separate addition to incubation medium strongly reduced the AA uptake. The influx of arachidonate was largely inhibited by ouabain and by incubation with mucosal sodium-free solution and amiloride, while it was increased when colonic mucosa was exposed to luminal amphotericin B. However, voltage-clamp studies showed that the AA entry rate appeared to be linearly related (r = 0.99) to transepithelial potential difference (PD) and suggested that the sodium dependence of AA translocation is an indirect effect of the changes in transepithelial PD induced by sodium transport shifts. These features provide evidence that there is a common entry pathway for AA and other long-chain free fatty acids mediated by a mechanism of facilitated diffusion driven by transmembrane PD.

To investigate on Na+, K+-ATPase behavior in chronic uremia, pre and postdialysis serum from 10 chronic dialysis patients and 10 healthy subjects was pooled and subjected to reverse phase C-18 HPLC. Only one fraction, isolated from pre and postdialysis sera, eluting at 28 min (F1), was found to display significant effects on electrophysiological and transepithelial 22Na flux pattern of rabbit distal colon mucosa mounted in Ussing type chambers; indeed, serosal addition of uremic F1 to colonic mucosa resulted in a slow, but constant, decline in short-circuit current (Isc) (deltaIsc = 1.55+/-0.16 microEq h(-1) cm(-2), mean +/- S.E.M., n=12, p0.01) and transepithelial conductance (G(T)) (from 4.50+/-0.23 to 3.71+/-0.33 mS cm(-2), p0.01, n=12). Measurement of transepithelial 22Na fluxes in the presence of pre or postdialysis sera also showed a significant Na+ absorption rate decrease (from 1.3+/-0.22 to 0.48+/-0.30 microEq h(-1) cm(-2), mean +/- S.E.M., n=4, p0.01), mainly due to a decrease in mucosal-to-serosal Na+ flux. By contrast, assays of peaks isolated from healthy sera did not inhibit Isc and transepithelial Na+ transport. The incubation of highly purified basolateral membranes with F1 for 1 min produced a approximately 26% inhibition of Na+, K+-ATPase. These findings are consistent with the presence of an endogenous inhibitor of sodium pump activity in uremic plasma; it is of pharmacological interest in that it may participate in the development of unpredictable responsiveness to digitalis therapy in pathophysiologic states.

Nitric oxide (NO) may play an important regulatory role in airway function. We have, thus, investigated in vitro whether epithelium derived NO may modulate cholinergic neurotransmission, via release of NO in guinea pig trachea, by using L-arginine (L-ARG), a precursor of NO synthesis, and L-N(G)-nitro-arginine-methyl-ester (L-NAME), an inhibitor of NO synthase. Results show that L-ARG and L-NAME modify acetylcholine sensitivity in epithelium-intact smooth muscle preparations, suggesting a probable NO synthesis by tracheal guinea pig epithelium.

RP610 hemofilters have been used up to five times in Post-dilutional hemofiltration. Clearance studies were Performed «in vivo» (creatinine and phosphate) and «in vitro» (Cr51 EDTA, I131 Hypaque, Co57 vitamin B12, H3 Inulin, C14 Dextran, l125 Albumin,) in new hemofilters and in those re-used once and five times. Hydraulic permeability and rejection coefficients, for the six markers different molecular weight, were also measured. Our preliminary results show that repeated cleansing with Amuchina® does not alter the characteristics of RP610 hemofilters. A scintigraphic method is suggested for visualizing possible changes in polarized areas between new and re-used hemofilters.

Effects of Ca2+ on adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion were investigated in intact mucosa and isolated crypt cells of rabbit descending colon. Addition of 10 microM prostaglandin (PG)E2 or forskolin to tissues incubated in Ca(2+)-free medium increased the size of short-circuit current (Isc) and Cl- secretion as estimated by unidirectional 36Cl flux measurements (net flux = -2.31 +/- 0.24 vs. -1.22 +/- 0.10 mueq.h-1.cm-2, n = 4, P < 0.001). Addition of 10 microM PGE2 to tissues incubated in 1.2 mM Ca2+ Ringer induced a 7-fold increase in mean cAMP level, whereas it produced an 11-fold increase in tissues exposed to Ca(2+)-free medium. Membrane preparations from whole mucosa incubated in Ca(2+)-free medium displayed a cyclic nucleotide phosphodiesterase activity significantly lower than controls (18.76 +/- 0.54 vs. 31.20 +/- 0.39 pmol cAMP. mg protein-1.min-1, means +/- SE, n = 4, P < 0.001). Ca2+ removal also affected adenylate cyclase (AC) responsiveness to agonists; AC activity increased in controls by 54 and 226% after stimulation with 10 microM PGE2 and forskolin, respectively, but it increased more (77 and 325%, respectively) after incubation in Ca(2+)-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationships between arachidonic acid (AA) metabolism and chloride secretion were investigated in mucosal preparations of rabbit distal colon. Tissues displayed a significant cyclooxygenase activity already in nonstimulated conditions and incubation with exogenous AA and calcium ionophore A23187 produced a predominant prostaglandin F2 alpha (PGF2 alpha) profile [PGF2 alpha greater than PGE2 greater than thromboxane B2 (TxB2) greater than 6-keto-PGF1 alpha] as assessed by HPLC of tissue homogenates, whereas 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) was not detected in AA- or A23187-stimulated tissues. Radioimmunological assays showed that PGE2 synthesis was time dependent, plateaued at 10 min, and proceeded at rates 15-20 times over TxB2 and 6-keto-PGF1 alpha. Among the PGs produced by colonic mucosa, only PGE2 and, to a lower extent, PGF2 alpha were found to stimulate chloride secretion and cAMP synthesis. Pretreatment with 10 microM 5,8,11,14-eicosatetraynoic acid, a cyclo- and lipoxygenase inhibitor, prevented AA-induced chloride secretion and PG and cAMP synthesis with the same strength as the cyclooxygenase inhibitor indomethacin. No effects were found after preincubation with nordihydroguaiaretic acid, a lipoxygenase blocker with moderate cyclooxygenase inhibitory properties, and caffeic acid, a lipoxygenase inhibitor. 5-HETE (5 microM) had no effect on short-circuit currents (Isc) and chloride transport, but it significantly reduced the increase in Isc, chloride secretion, and PGE2 synthesis elicited by AA or A23187. Platelet-activating factor, reported to stimulate rabbit colon Isc through an indomethacin-sensitive pathway, was not detected at concentrations as low as 10(-10) M.