C. Lippe

Actions of vasopressin and isoprenaline on the ionic transport across the isolated frog skin in the presence and the absence of adenyl cyclase inhibitors MDL12330A and SQ22536.

1. The effects of both adenyl cyclase inhibitors (MDL12330A and SQ22536) have been studied on the ionic transport induced by vasopressin and isoprenaline across the frog skin. 2. MDL12330A inhibits the vasopressin action on the short-circuit current (SCC), confirming that this effect is cAMP-mediated. 3. On the other hand, isoprenaline action on the SCC is unaffected by MDL12330A. However, this lack of effect is not a sufficient argument against the role of cAMP in this action; in fact, as MDL12330A is also an inhibitor of cAMP phosphodiesterase, this action could mask the inhibitory effect of the drug on adenyl cyclase. 4. By using the other adenyl cyclase inhibitor (SQ22536), probably deprived of effect on the cAMP phosphodiesterase, we obtained a strong inhibition of isoprenaline action on the SCC. Thus we conclude that the actions of isoprenaline on the ionic transport across the frog skin are also cAMP-mediated.

Why Amphibians Are More Sensitive than Mammals to Xenobiotics

Dramatic declines in amphibian populations have been described all over the world since the 1980s. The evidence that the sensitivity to environmental threats is greater in amphibians than in mammals has been generally linked to the observation that amphibians are characterized by a rather permeable skin. Nevertheless, a numerical comparison of data of percutaneous (through the skin) passage between amphibians and mammals is lacking. Therefore, in this investigation we have measured the percutaneous passage of two test molecules (mannitol and antipyrine) and three heavily used herbicides (atrazine, paraquat and glyphosate) in the skin of the frog Rana esculenta (amphibians) and of the pig ear (mammals), by using the same experimental protocol and a simple apparatus which minimizes the edge effect, occurring when the tissue is clamped in the usually used experimental device. The percutaneous passage (P) of each substance is much greater in frog than in pig. LogP is linearly related to logKow (logarithm of the octanol-water partition coefficient). The measured P value of atrazine was about 134 times larger than that of glyphosate in frog skin, but only 12 times in pig ear skin. The FoD value (Pfrog/Ppig) was 302 for atrazine, 120 for antipyrine, 66 for mannitol, 29 for paraquat, and 26 for glyphosate. The differences in structure and composition of the skin between amphibians and mammals are discussed.

Polar distribution of sodium-dependent and sodium-independent transport system forl-lactate in the plasma membrane of rat enterocytes

The uptake ofl-lactate by rat small intestinal brush-border and basal-lateral plasma membrane vesicles has been studied.l-Lactate uptake by the isolated membrane vesicles is osmotically sensitive and represents predominantly transport into an intravesicular space and not binding to the membranes.The transport ofl-lactate across the brush-border membrane is stimulated by sodium, whereas the transport across the basal-lateral plasma membrane is sodium-independent. In both types of membrane vesiclesl-lactate is transported faster thand-lactate andl-lactate transport is inhibited by α-cyano-cinnamic acid.l-Lactate transport across basal-lateral membranes is inhibited byd-lactate and pyruvate and transstimulated byl-lactate and pyruvate.The polar distribution of transport system forl-lactate in the plasma membrane of rat enterocytes—a Na+/l-lactate cotransport system in the brush-border membrane and a facilitated diffusion system in the basal-lateral membrane — can explain the fact that in the intact epitheliuml-lactate produced by cell metabolism is preferentially released on the serosal side and could enable the cell to perform vectorial, secondary active transport ofl-lactate from the intestinal lumen to the serosal compartment.

Effect of prostaglandin E2 on the proliferation, Ca2+ mobilization and cAMP in HT-29 human colon adenocarcinoma cells

Abstract Several lines of evidence suggest that non-steroidal antiinflammatory drugs (NSAIDs) have anticarcinogenic effects. The causal relationship linking the preventive effect of NSAIDs on colon cancer and the inhibition of prostaglandin synthesis is questioned by the contrasting results obtained by many laboratories. The experiments reported in this paper demonstrate that prostaglandin E 2 (PGE 2 ) did not stimulate the proliferation in HT-29 human colon adenocarcinoma cells under several experimental conditions. Moreover, PGE 2 and 17-phenyl trinor prostaglandin E 2 (a specific agonist of EP1 receptors) did not increase intracellular Ca 2+ concentration. Finally, PGE 2 did not affect the intracellular cAMP and did not reduce the isoproterenol dependent increase in cAMP. These results indicate that in HT-29 cells: (1) proliferation is not directly sensitive to PGE 2 ; and (2) PGE 2 does not stimulate a signal transduction pathway leading to intracellular increase in cAMP or Ca 2+ mobilization. Therefore, other cell lines should be used to assess the direct role played by prostanoids in promoting cell proliferation in colon cancer.

The proliferative response of HT-29 human colon adenocarcinoma cells to bombesin-like peptides.

Bombesin-like peptides (BLP) and their receptors are widely distributed throughout the intestine and are potential mitogens for gastrointestinal cancers. In this study we characterized the proliferation induced by BLP in the human adenocarcinoma cell line HT-29. The number of HT-29 cells, partially serum deprived (1% fetal bovine serum) for 48 h, was increased after 24 h of stimulation with bombesin, GRP, neuromedin B (NMB) and neuromedin C (NMC) ranging from 0.1 nM up to 1 microM. Reverse transcription polymerase chain reaction studies, revealed the presence of mRNA for NMB and for the GRP preferring receptor (GRP-R). mRNA for GRP, NMB preferring receptor (NMB-R) and bombesin receptor subtype 3 (BRS-3) were not detected. [D-Phe(6)]bombesin-(6-13)methyl ester (A1) and BIM-23127 (A2), are considered as inhibitors of binding to GRP-R and NMB-R, respectively. Surprisingly, A1 and A2 stimulated the proliferation of HT-29 cells. Moreover, in the simultaneous presence of 1 microM A1 and 0.1 microM GRP or 0.1 nM or 0.1 microM bombesin, inhibition of the proliferation was observed. Our data demonstrate that the proliferation induced by BLP in HT-29 cells is due to interaction with the GRP-R.

Atrazine increases the sodium absorption in frog (Rana esculenta) skin

The presence of atrazine in agricultural sites has been linked to the decline in amphibian populations. The efforts of the scientific community generally are directed toward investigating the long-term effect of atrazine on complex functions (reproduction or respiration), but in the present study, we investigated the short-term effect on the short-circuit current (I(sc)), a quantitative measure of the ion transport operated by frog (Rana esculenta) skin. Treatment with 5 microM atrazine (1.08 mg/L) does not affect the transepithelial outfluxes of [14C]mannitol or [14C]urea; therefore, atrazine does not damage the barrier properties of frog skin. Atrazine causes a dose-dependent increase in the short-circuit current, with a minimum of 4.64 +/- 0.76 microA/cm2 (11.05% +/- 1.22%) and a maximum of 12.7 +/- 0.7 microA/cm2 (35% +/- 2.4%) measured at 10 nM and 5 microM, respectively. An increase in Isc also is caused by 5 microM ametryne, prometryn, simazine, terbuthylazine, or terbutryn (other atrazine derivatives). In particular, atrazine increases the transepithelial 22Na+ influx without affecting the outflux. Finally, stimulation of Isc by atrazine is suppressed by SQ 22536, H89, U73122, 2-aminoethoxydiphenyl borate, and W7 (blockers of adenylate cyclase, protein kinase A, phospholipase C, intracellular Ca2+ increase, and calmodulin, respectively), whereas indomethacin and calphostin C (inhibitors of cyclooxygenase and protein kinase C, respectively) have no effect.

Facilitated transport of urea across the toad gallbladder.

SummaryThe toad gallbladder epithelium is much more selective than that of the rabbit expecially as to the permeability of two molecules like urea and thiourea.These observations can probably be attributed to different permeation mechanisms of the 2 molecules.Neither active transport nor solvent drag can explain these phenomena.10−4 M phloretin strongly inhibits urea movement, but does not alter either thiourea fluxes or isotonic net water transport: these results suggest that a specific mechanism is involved in urea movement.The urea transport shows saturation kinetic which is consistent with the presence of a facilitated mechanism.

Action of capsaicin and related peptides on the ionic transport across the skin of Rana esculenta

Capsaicin at low concentrations increases the short circuit current (SCC) across frog skin. Simultaneous measurements of both transepithelial fluxes of 22Na or 36Cl demonstrate that the SCC increase is due to stimulation of sodium active absorption. Capsaicin acts through the liberation of several peptides; thus these peptides were tested on the SCC across frog skin. Those more active are, in order of potency: Cyclic Calcitonin Gene Related Peptide (CGRP), Kassinin and Eledoisin, Substance P (SP) and Neurokinin A. Neurokinin B and Vasoactive Intestinal Peptide (VIP) have no effect. Also the actions of SP and CGRP are due mainly to stimulation of Na+ active absorption. A strict parallelism regarding the sensitivity to inhibitors (Naproxen, SQ22536 and CP96345) between SP, CGRP and Capsaicin strengthens the hypothesis that SP and CGRP are liberated by Capsaicin in this tissue.

Actions of carbaryl on the ionic transport across the isolated skin of Rana esculenta.

1. Carbaryl, a carbamate used as a pesticide, increases the short-circuit current (SCC) across the isolated frog skin in a dose-dependent manner. 2. This effect is due to the stimulation of sodium absorption and chloride secretion. 3. Carbaryl action on short-circuit current is unrelated to its inhibitory power on cholinesterase; this statement is supported by two experimental results: (a) carbaryl is equally active on both sides of the skin, (b) atropine pretreatment does not inhibit the carbaryl action on SCC.

Amide and thio-amide permeability across the urinary bladder of Rana esculenta. Effect of posthypophyseal hormones

Abstract 1. 1. The permeability of several amides and thio-amides across the isolated urinary bladder of Rana esculenta has been studied. 2. 2. The urinary bladder presents, at least qualitatively, the same permeability sequence of amides of artificial phospholipid membranes. 3. 3. The addition of neurohypophyseal hormones results in a large increase of amide permeability. In these conditions the urinary bladder does not present the permeability sequence previously reported. 4. 4. Contrary to what has been found by Maffly et al. (1960) in Bufo marinus bladder, the R. esculenta bladder under ADH treatment shows a slight and delayed increase of permeability to thiourea and related compounds. 5. 5. Most probably there exists a different pathway for amide and thio-amide permeability through the frog urinary bladder.