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.

The adrenergic receptor subtypes present in frog (Rana esculenta) skin.

Frog skin transports ions and water under hormonal control. In spite of the fundamental role played by adrenergic stimulation in maintaining the water balance of the organism, the receptor subtype(s) present in the skin have not been identified yet. We measured the increase in short-circuit current (ISC, an estimate of ion transport) induced by cirazoline, clonidine, xamoterol, formoterol, or BRL 37344, in order to verify the presence of alpha1, alpha2, beta1, beta2, or beta3 receptor subtypes, respectively. Only after treatment with formoterol, BRL 37344 and, to a lesser extent, cirazoline was measured a significant increase in ISC (57%, 33.2%, and 4.7%, respectively). The formoterol and BRL 37344 concentrations producing half-maximal effect (EC50) were 1.12 and 70.1 nM, respectively. Moreover, the formoterol effect was inhibited by treatment with ICI 118551 (antagonist of beta2 receptors) while SR 59230A (antagonist of beta3 receptors) had no effect; opposite findings were obtained when the BRL 37344 stimulation was investigated. Finally, by measuring the transepithelial fluxes of 22Na+ and 36Cl-, we demonstrated that Na+ absorption is increased by activation of beta2 and beta3 and is cAMP-sensitive, whereas the Cl- secretion is only increased by activation of beta2 receptors and is cAMP- and calmodulin-sensitive.

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.

Eledoisin and Kassinin, but not Enterokassinin, stimulate ion transport in frog skin.

In frog skin, tachykinins stimulate the ion transport, estimated by measuring the short-circuit current (SCC) value, by interacting with NK1-like receptors. In this paper we show that Kassinin (NK2 preferring in mammals) increases the SCC, while Enterokassinin has no effect. Therefore, either 2 Pro residues or 1 Pro and 1 basic amino acid must be present in the part exceeding the C-terminal pentapeptide. Eledoisin (NK3 preferring in mammals) stimulation of SCC is reduced by CP99994 and SR48968 (NK1 and NK2 antagonists) and not affected by SB222200 (NK3 antagonist). None of the three antagonists affects Kassinin stimulation of SCC.

Inositol 1,4,5-trisphosphate and ryanodine receptors mobilize calcium from a common functional pool in human U373 MG cells.

This investigation concentrates on the change in Ca(2+) concentration ([Ca(2+)]) caused by ryanodine in U373 MG cells. This cell type from a human astrocytoma is a unique cellular model because it only expresses the type 3 ryanodine receptor (RyR3), which is generally the least abundant isoform. In the presence of physiological [Ca(2+)] in the extracellular medium, U373 MG cells are caffeine-insensitive, even after forskolin treatment, and ryanodine-sensitive only when an unusually high concentration (30 microM) is applied. Xestospongin C behaves like thapsigargin and therefore cannot be used as a selective antagonist of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). After ryanodine challenge, addition of an analog of Substance P (SP), which should deplete InsP(3)-sensitive stores, has no effect on [Ca(2+)](i). After thapsigargin treatment, which unmasks the calcium leak from intracellular stores, neither ryanodine nor SP change [Ca(2+)](i), suggesting that thapsigargin completely depletes the ryanodine-sensitive and the InsP(3)-sensitive stores of U373 MG cells. Finally, in experiments monitoring the [Ca(2+)] in intracellular stores, InsP(3) stimulation of permeabilized cells causes a decrease in [Ca(2+)] that is not affected by subsequent ryanodine treatment. Our results support the conclusion that U373 MG cells express both InsP(3)Rs and RyRs that can individually or in combination mobilize only one functional Ca(2+) pool.

Calcium response after stimulation by substance P of U373 MG cells: inhibition of store-operated calcium entry by protein kinase C.

In this paper we investigate the Ca2+ response after Substance P (SP) stimulation of U373 MG cells. SP is a tachykinin and physiologically acts as a neurotransmitter and neuromodulator in the nervous system, but pathologically triggers malignant glial cells, such as U373 MG, to release cytokines and increase proliferation rate. In this paper we show that SP increases the proliferation rate of U373 MG cells and the intracellular Ca2+ concentration by mobilizing Ca2+ only from thapsigargin-sensitive stores. In fact, Ca2+ entry through store-operated calcium entry (SOCE) channels, which was observed after thapsigargin treatment, was not detected after stimulation by SP. The inhibition of SOCE after SP stimulation must be mediated by protein kinase C (PKC), because it was not observed in the presence of calphostin C (an inhibitor of PKC). Moreover, stimulation by SP-induced membrane potential hyperpolarization. Our results are consistent with the following sequence of events: (i) SP interacts with NK(1) receptors; (ii) fast homologous receptor desensitization occurs; (iii) reuptake by endoplasmic reticulum Ca(2+)-ATPase quantitatively overwhelms the extrusion by plasma membrane Ca2+-ATPase. These results have two important consequences. In U373 MG cells the SOCE does not contribute to the Ca2+ response after SP, and is not necessarily involved in promoting cell proliferation.

Pyrethroid stimulation of ion transport across frog skin.

Pyrethroids are grouped into two classes (types I and II) because of the absence or presence of an alpha-cyano substituent and the production of a different intoxication syndrome in rodents. In this study, we investigated the effect of pyrethroids on the ion transport across frog skin (Rana esculenta). The short-circuit current value (estimate of ion transport) was increased by each of the eight pyrethroids tested, with the following order of potency: lambda-cyhalothrin > deltamethrin > alpha-cypermethrin = beta-cyfluthrin > bioallethrin > permethrin > bioresmethrin > phenothrin. The first four compounds are type II pyrethroids. Therefore, ion transport is stimulated more by type II pyrethroids than by type I. Experiments performed in the presence of amiloride support the conclusion that pyrethroids mainly increase Na+ absorption and to a lesser extent Cl- secretion. In these experiments, no systematic difference between type I and II pyrethroids was found. Finally, the stimulation by pyrethroids was inhibited by indomethacin and W7 (inhibitors of cyclooxygenases and the Ca2+/calmodulin system, respectively). These observations suggest that pyrethroids do not directly affect the epithelial Na+ channel (ENaC) but indirectly influence an intracellular event involved in ENaC modulation and linked to the Ca2+ signaling cascade.