Epithelial and mucosal preparations from canine colon: responses to substance P.

The hypothesis that substance P (SP) elicits both direct and indirect responses on the canine proximal colon was tested using two different in vitro preparations. The mucosa contained the muscularis mucosa and the attendant submucosal plexuses whereas the epithelium, being devoid of both, was functionally "nerve-free." Dose-dependent stimulation was noted on both preparations, increases in peak current (microamperes per squared centimeter) as well as charge transfers (millicoulombs per squared centimeter) were monitored. Tetrodotoxin significantly reduced mucosal responses whereas atropine and the H1 antagonists mepyramine and diphenhydramine had only marginal effects. None of these agents affected the responses of the epithelium to SP. Indomethacin significantly reduced responses in both preparations. Removal of Na+ or Cl- or the use of C- channel blockers (9-anthracene carboxylic acid and N-phenylanthranilic acid) produced a significant reduction of SP responses across the epithelium. Thus, SP has both direct and indirect affects on the colon; activation of the cyclooxygenase pathway could be significant.

An "epithelial" preparation from the canine gastric mucosa: responses to secretagogues.

Isolated mucosal preparations from the stomach are generally obtained by removing the external muscle layers, leaving intact the muscularis mucosae. By carefully dissecting off the muscularis mucosa, we have obtained "epithelial" preparations from the canine stomach. Such preparations responded to secretagogues, histamine, pentagastrin, and a cholinergic agonist (cis-2-methyl 4-dimethylaminomethyl 1,3-dioxolane methiodide). Epithelial preparations were, in general, more sensitive than mucosal preparations. Of the three secretagogues tested, histamine was the most consistent stimulant, with well-sustained responses being noted. Responses to the other agonists were more variable. Pentagastrin produced brief but explosive increases in acid output.

Luminal receptors for bradykinin on the canine tracheal epithelium: functional subtyping.

Luminal addition of bradykinin (BK) to the open-circuited canine tracheal epithelium produces a biphasic response in transmucosal potential difference (P.D.): a rapid, transient decrease (dip) followed by a subsequent, more sustained increase (rise), both phases being associated with an increase in conductance. We have attempted to characterise the receptor subtype mediating the bradykinin response. Lys-bradykinin (Lys-BK) elicited a similar response, and its EC50 as judged from concentration-response relations was similar to that of BK. Cross-tachyphylaxis between the two peptides confirmed a common receptor. Des-Arg9-BK (a B1-agonist) neither elicited a response nor inhibited responses to BK. The novel B2-antagonist [Thi6,9-D-Phe8]kallidin reversibly inhibited responses to both BK and Lys-BK. The rapid changes in P.D. (dips) were unaffected by Na+ removal, but were eliminated by replacing luminal Cl- with isethionate. Thus, BK, acting on B2-receptors, transiently increases anion permeability of the luminal membrane of the canine tracheal epithelium.

Effects of TMB-8, a calcium antagonist, on transport properties of the isolated canine tracheal epithelium.

The effects of the putative intracellular Ca2+ antagonist, TMB-8 (8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate), on the canine tracheal epithelium were examined. Luminal addition reduced rapidly, but reversibly, the transmucosal potential difference and increased the resistance across the open-circuited epithelium. Under short-circuit conditions, the drug reduced stimulation by prostaglandin E2, forskolin, 8-bromo cyclic AMP, prostaglandin F2 alpha and A23187. Inhibition of prostaglandin E2 responses were accompanied by reversal of net Cl- fluxes produced by the agonist. The effects of TMB-8 were unaffected by increasing Ca2+ in the bathing solutions, and were not mimicked by procaine, nitrendipine, calmidazolium, compound 48/80 or trifluoperazine. W7 did, to a limited extent, produce similar responses, though the drug was more toxic, and the effects were irreversible.

Luminal tachykinin receptors on canine tracheal epithelium: functional subtyping.

Luminal addition of tachykinins to the open-circuited canine tracheal epithelium produces a biphasic response in the transmucosal potential difference (PD). A rapid, transient decrease is followed by a subsequent rise, both phases being associated with changes in conductance. Concentration-response curves demonstrated the following orders of potency: substance P greater than physalaemin greater than eledoisin = kassinin for the tachykinins, and substance P greater than substance P-(4-11) greater than substance P-(6-11) using the C-terminal fragments. Both sequences are similar to those reported for the dog carotid artery. These observations were confirmed by cross-tachyphylaxis experiments. SP-O-methyl ester, a selective agonist for the SP-P (or NK-1) receptor, elicited identical responses, and exhibited cross-tachyphylaxis to substance P. Bradykinin produced similar luminal responses, though different receptors are involved, since no cross-tachyphylaxis was observed between bradykinin and the tachykinins.

Simultaneous measurements of 22Na and 36Cl in aqueous samples: a comparison of three different methods.

We have critically analyzed three methods that measure the combined presence of 36Cl and 22Na in aqueous samples. These were a sequential method, a dual-label spectral analysis method (Beckman 1983), and a AgNO3 precipitation method (Thompson 1983). The former requires the use of both a gamma counter and a beta liquid scintillation counter, whereas the latter two require access to a beta counter only. Our analysis suggests that investigators who have access to both a liquid scintillation counter and a gamma spectrometer would find the sequential method relatively simple to use and reasonably reliable. Those with access to only a liquid scintillation counter would be best advised to use the AgNO3 method described by Thompson.

Histamine stimulation of canine colonic epithelium: potentiation by hydroxylamines.

Hydroxylamine derivatives markedly potentiated responses of the canine colonic mucosa to histamine, but not to carbachol or 5HT. Effects noted were produced upon either luminal or serosal addition of the agents, suggesting an intracellular mechanism. Similar potentiations were observed with a phosphodiesterase inhibitor, IBMX, suggesting that hydroxylamines could act by altering cyclic nucleotide levels. Since such derivatives could be produced as intermediates by colonic bacteria, the effects noted could have pathological implications.

Peptides increase anion conductance of canine trachea: an effect on tight junctions.

The ionic basis for the rapid reduction in potential difference (dip) produced on luminal addition of substance P and related peptides was analysed by altering the electromotive force and chemical gradients across the isolated, canine tracheal epithelium. The dip could be exaggerated, minimised or reversed by increasing, decreasing or reversing the basal potential difference, and the intercept of the line relating the two was close to zero when the Cl- compositions of the two bathing solutions were identical. Luminal Cl- replacement by a non-permeant anion (isethionate) attenuated the dip which was, however, exaggerated by a permeant anion (nitrate). Replacement of serosal Na+ or luminal HCO3- had no significant effect on the magnitude of the dip. The tachykinins exhibited cross-tachyphylaxis with each other, indicating a common receptor. Bradykinin, a structurally unrelated peptide, also produced dips upon luminal addition, but showed no cross-tachyphylaxis with the tachykinins. Again, a linear relation between basal potential difference and the dip elicited by bradykinin was observed. Based on current awareness of the bioelectric properties of the canine tracheal epithelium, we suggest that these peptides modulate paracellular anion permeabilities.

Epithelial and mucosal preparations of canine proximal colon in ussing chambers: comparison of responses.

A novel epithelial preparation from proximal canine colon, devoid of its underlying muscularis mucosa, was studied in Ussing chambers and its responses to added agonists compared with those of a conventional full mucosal preparation. We assumed that the responses elicited from the former would reflect direct action of agonists on epithelial cells, and from the latter both direct and indirect effects mediated by functioning nerves in the attached submucosal plexus. Responses of the two preparations to tetrodotoxin, ouabain, scorpion venom, and field stimulation were different, the differences being explicable on the basis of functioning nerves in the mucosal preparation. Histamine stimulated both preparations; the increase in short-circuit current seen with the mucosal but not the epithelial preparation was inhibited by tetrodotoxin. Serotonin was a better stimulant of the epithelial preparation. Basal absorptions of both Na+ and Cl- were seen in both mucosa and epithelium, but net absorptions were significantly greater in the latter, suggesting tonic neuronal inhibition of absorption in the mucosa. These two preparations would prove useful in analysing direct and indirect effects of agonists on colonic transport.

Ouabain-insensitive, halide-sensitive Tl+ uptake by canine iliac arteries.

Ouabain-insensitive Tl+ uptake by canine iliac arteries was studied. A significant fraction was halide-sensitive, with Br- substituting well for Cl-. The halide-sensitive component was inhibited by diuretics (MK196, bumetanide), PCMBS, low temperatures and external cations K+, Rb+. External but not internal Na+ was necessary for the uptake process. The process was not sensitive to disulphonic stilbenes. The halide-sensitive uptake appears to represent the operation of a (Na+/K+/Cl-)-cotransport process in arteries.

Effects of tachykinins on the electrical activity of isolated canine tracheal epithelium: an exploratory study.

Substance P (SP) and other tachykinins altered the potential differences (P.D.) and resistances (omega) of open-circuited epithelial preparations. (1) The effects observed were critically dependent on the side to which the peptides were added. Luminal addition of SP (5 x 10(-7) M) produced within 8-20 s, a rapid decrease in P.D. (dip) followed by an increase that peaked transiently and declined. Serosal addition led to an increase in P.D. after a longer lag (40-90 s). In both cases, resistance decreased. (2) Low concentrations of SP (5 x 10(-12) M) elicited only an increase in P.D., the dip appearing at concentrations 50-100-fold higher, indicating perhaps receptors with different affinities. (3) Changes in P.D. and resistance were seen on luminal addition of physalaemin, eledoisin, kassinin, alpha-neurokinin, neuromedin K and C-terminal SP fragments larger than 5 amino acids. No responses were seen with SP tetrapeptide, SP 9-11, bombesin, litorin, neurotensin, dynorphin. The sequence Phe-X-Gly-Leu-Met-NH2 thus seems necessary to elicit changes in P.D. and resistance. (4) As with SP, low doses of physalaemin, eledoisin, kassinin elicited only an increase in PD, the dip appearing with higher concentrations.

The sodium pump in opossum vascular smooth muscle.

Ouabain-sensitive Rb+ uptake and [3H]ouabain binding were used to measure rates of Na+ pumping and the number of pump sites, respectively, in thoracic aortae from opossums. From the number of Rb+ ions pumped per site per minute, estimates of pump turnover have been made. Values obtained are comparable to those of other species (see Table 1).

Intrinsic nervous control of migrating myoelectric complexes.

The role of intrinsic nerves in the control of migrating myoelectric complexes (MMCs) was studied in seven conscious dogs, each implanted with a set of eight bipolar Trimel wire electrodes. Local areas, 3-5 cm long, were perfused close intra-arterially via an exteriorized heparinized Silastic cannula. Experiments consisted of giving bolus injections of atropine (20-50 micrograms), hexamethonium (20 mg), and tetrodotoxin (TTX; 3-30 micrograms) via the catheter at varying periods of time with respect to the arrival of phase III at the perfused site. Atropine and hexamethonium, given close intra-arterially immediately before the arrival of phase II at the perfused site, blocked its further propagation. Tetrodotoxin given locally also blocked the propagation of phage III, as above. After the block, TTX initiated a new phase III activity at, or distal to, the perfused site in 10 out of 14 perfusions. The new phase III activity propagated distally. This study shows that the mechanisms for the initiation and propagation of MMCs are built into the enteric plexus. Once an MMC is initiated, its propagation is achieved by proximal-to-distal excitation through the intrinsic cholinergic network of neurons. This study explains the lack of any significant changes in the propagation parameters of MMCs after vagotomy or celiac and superior mesenteric ganglionectomy.