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Electrophysiological responses of frog skin to the peptides bombesin, caerulein, and physalaemin
Peptides 20 (1999) 1239 –1241
Short communication
Electrophysiological responses of frog skin to the peptides bombesin, caerulein, and physalaemin Daniel F. Stiffler* Biological Sciences Department, California State Polytechnic University, Pomona, CA 91768, USA Received 13 April 1999; accepted 2 June 1999
Abstract Isolated skins from the frog Rana pipiens were mounted on Ussing-type chambers and bathed with Amphibian Ringer’s solution on both sides. Electrical potential difference, resistance, and short-circuit current (SCC) were measured by using calomel electrodes, Ag-AgCl electrodes, Ringer’s-agar bridges, and Tektronix digital multimeters. Under the conditions employed, SCC is a measure of net Na⫹ transport. The frog skin peptides bombesin, caerulein, and physalaemin were administered to the serosal side at concentrations of 0.5, 5.0, and 50 ng/ml. Control electrophysiological parameters were: potential difference, 23 ⫾ 2 mV; resistance, 738 ⫾ 59 ⍀ cm2; and SCC 32 ⫾ 3 A/cm2. Although bombesin and caerulein had no significant, reversible effect on potential difference, resistance, or SCC, physalaemin significantly, and reversibly, depressed SCC by 22%. Caerulein did significantly depress SCC, but the response was not reversible. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Amphibian; Rana pipiens; Bioactive peptides of the skin; Cutaneous Na⫹ transport
1. Introduction Frog skin produces a wide variety of polypeptides whose structure, but not function, in amphibians, is well known. There are several groups of these peptides. One, the tachykinins, is represented by a mammalian brain peptide known as substance-P. A frog-skin peptide that shows sequence homology with substance-P comes from the frog Physaleamis fusculamatus and is called physalaemin [5]. A second group is called the caeruleins. The first of these to be isolated was caerulein from Hyla caeruleia [2]. Caerulein is related to the mammalian hormone cholecystokinin-pancreozymin [4]. A third group is called the bombesin-like peptides. Bombesin was first isolated from the skin of Bombina bombina [1]. It was not until after the discovery of bombesin in amphibian skin that a mammalian counterpart— gastrin-releasing peptide—was discovered [4]. Although there are data describing actions of these peptides in mammalian systems, there is very little on their actions in frog skin. Transport of water and electrolytes are two of the important physiological functions of amphibian * Corresponding author. Tel.: ⫹1-909-869-4061; fax: ⫹1-909-8694078. E-mail address: [email protected] (D.F. Stiffler)
skin that might be modulated by one or more of the peptides. When placed on the outside (mucosal) surface of Rana pipiens skin, caerulein (pmolar concentrations) caused an increase in short-circuit current (SCC) that was interpreted to result from increased net Na⫹ transport across the skin [9]. Physalaemin had no effect on water permeability when used alone on the serosal side of the toad bladder; it was capable of abolishing the hydroosmotic responses to vasopressin and oxytocin [7]. Bombesin may stimulate Na⫹ transport in the mammalian kidney [6]. The objectives of the present study were to evaluate the serosal effects of bombesin, caerulein, and physalaemin on electrical potential difference (PD), SCC, and resistance (R) in the skin of the frog R. pipiens.
2. Materials and methods Frogs (R. pipiens) were purchased from commercial suppliers. The frogs were pithed, and a piece of ventral skin was mounted on one end of a glass cylinder. A plastic cap with an opening equal to the diameter of the opening in the glass cylinder (3.14 cm2) was constructed to fit over the piece of frog skin that was stretched over the end of the
0196-9781/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 9 9 ) 0 0 1 2 8 - X
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D.F. Stiffler / Peptides 20 (1999) 1239 –1241
Fig. 1. Frog-skin SCC responses to to serosal bombesin (open circles), caerulein (closed circles), and physalaemin (open triangles). The numbers of observations are in parentheses. * Physalaemin-treated skins were significantly different than controls at a dose of 50 ng/ml (P ⬍ 0.05; Tukey’s test for multiple comparisons). When the physalaemin was removed, the SCC significantly reversed to control levels (P ⬍ 0.05; Tukey’s test). The results of one-way analysis of varience were F(4,35) ⫽ 4.18; P ⫽ 0.0072. Bombesin-treated skins appeared to decrease SCC but the results were not significant [F(4,30) ⫽ 1.54]; P ⫽ 0.2166. Caerulein, like physalaemin, caused a significant decrease in SCC (*) at 50 ng/ml (P ⬍ 0.05; Tukey’s test) but unlike with physalaemin the decrease was not reversible. The results of one-way ANOVA were F(4,35) ⫽ 3.19; P ⫽ 0.0247.
glass cylinder (serosa in). An o-ring was placed inside the plastic cap to seal the skin tightly against the glass, and silicone grease was used on both sides of the skin to increase the seal. The plastic cap was held in place with rubber bands that attached to hooks on both the cylinder and cap. The cylinder with frog skin was filled with Ringer’s solution (composition: NaCl, 6.5 g/l; KCl, 0.14 g/l; CaCl2, 0.12 g/l; NaHCO3, 1.26 g/l). The Ringer’s filled cylinder apparatus then was suspended in Ringer’s solution so that there was no pressure gradient across the skin. PD-electrodes (Ringer’s-3% agar bridges) were attached to calomel electrodes that were attached to the input poles of a Tektronix DM 502 multimeter (Beaverton, OR, USA). SCC electrodes (saturated KCl-agar bridges) were placed with their ends opposite the center of the skin in both chambers. These current-passing bridges were connected to another DM 502 multimeter via Ag-AgCl electrodes. A 90-V dry cell battery and potentiometer were inserted in the circuit to null the spontaneous potential difference exhibited by the frog skin. The current required to accomplish this is the SSC and is equal to net Na⫹ flux under these conditions [11]. The ratio of the PD and SCC is the electrical resistance (⍀ cm2) that is directly related to ionic permeability. Once the skins were mounted, the potential difference was allowed to stabilize (1 h) before the experiments were started. The peptides were added to the Ringer’s solution in the cylinder (serosa) so that the final concentration in the cylinder equilibrated at 0.5, 5.0, and 50.0 ng/ml for approximately 20 min at each concentration. After the final concentration was tested, the chamber was flushed and refilled
with fresh Ringer’s solution. The peptides were purchased from Sigma (St. Louis, MO, USA).
3. Results and discussion The mean PD for the skins was 23 ⫾ 2 mV. The mean SCC was 32 ⫾ 3 A/cm2. The mean R was 738 ⍀ cm2. Of the three peptides, physalaemin had the greatest effect. Serosal treatment produced a decrease in SCC (Fig. 1), becoming significant (P ⬍ 0.05) at 50 ng/ml. Removal of the peptide resulted in a clear recovery (P ⬍ 0.05) as SCC (Fig. 1) returned to pre-control values. Caerulein, applied to the serosal surface, also produced a significant (P ⬍ 0.05) decrease in SCC; however, this decrease did not clearly reverse when the caerulein was removed. Although the recovery SCC was not significantly different than the 50 ng/ml treatment SCC, it was also not significantly different than the control SCC (0.05 ⬍ P ⬍ 0.1). Neither caerulein nor physalaemin caused significant changes in PD or R. Bombesin, applied to the serosal surface, had no significant effect on PD, SCC, or R. Physalaemin-like tachykinins have been detected in the blood of R. pipiens at 1.1–2 ng/g [3]. Circulating values for bombesin and caerulein have not been published; however, effective doses range from ng/kg to g/kg in a wide variety of systems [4]. The decrease in SCC induced by physalaemin and perhaps caerulein may have resulted from a decrease in active Na⫹ transport. Amphibian skin has been reported to contain highly polar substances that inhibit Na⫹, K⫹-ATPase [8]. Serosal applications of caerulein bring about different
D.F. Stiffler / Peptides 20 (1999) 1239 –1241
responses (inhibition of SCC) than do mucosal applications that stimulate SCC [9]. Caerulein interacts with dopaminergic neurons [4], which may explain this difference. Dopamine inhibits Na⫹ transport in frog kidney tubules [10].
References [1] Anastasi A, Erspamer V, Bucci M. Isolation and amino acid sequence of alytesin and bombesin, two analogous tetradecapeptides from the skin of European discoglossid frogs. Arch Biochem Biophys 1972; 148:443– 6. [2] Anastasi A, Erspamer V, Endean R. Isolation and amino acid sequence of caerulein, the active peptide in the skin of Hyla caerulea. Arch Biochem Biophys 1968;125:57– 68. [3] Creagh T, Skrabanek P, Cannon D, Balfe A, Powell D. Phylogeny of substance P. Gen Comp Endocrinol 1980;40:503– 6. [4] Erspamer V. Bioactive secretions of the integument. In: Heatwole H, Barthalmus GT, editors. Amphibian Biology, The Integument, Vol. 1. Chipping Norton, Australia: Surrey Beatty & Sons, 1994. pp. 179 –350.
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[5] Erspamer V, Anastasi A, Cei JM. Structure and pharmacological actions of physalaemin, the main active polypeptide of the skin of Physalaemis fuscumaculatus. Experientia 1964;20:489 –90. [6] Erspamer V, Melchiorri P, Sopranzi N. The action of bombesin on the kidney of the anesthetized dog. Br J Pharmacol 1973;48:438 – 55. [7] Furtado MFR. Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder. Effects of bradykinin, kallidin, eledoisin, and physalaemin. J Memb Biol 1971;4:165–78. [8] Flier J, Edwards MW, Daly JW. Widespread occurrance in frogs and toads of skin compounds interacting with ouabain sites and Na⫹, K⫹-ATPase. Science 1980;208:503–5. [9] Greenwell JR, Low HS. Action of caerulein, gastrin 17, pentagastrin, and secretin on active transport of sodium by frog Rana pipiens skin. J Memb Biol 1981;61:91– 6. [10] Hagiwara N, Kubota T, Kubakawa M, Fujimoto M. Effects of dopamine on the transport of Na, H, and Ca in the bullfrog kidney tubule. Jpn J Physiol 1990;40:351– 68. [11] Ussing HH, Zerahn K. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol Scand 1951;23:110 –27.
Short communication
Electrophysiological responses of frog skin to the peptides bombesin, caerulein, and physalaemin Daniel F. Stiffler* Biological Sciences Department, California State Polytechnic University, Pomona, CA 91768, USA Received 13 April 1999; accepted 2 June 1999
Abstract Isolated skins from the frog Rana pipiens were mounted on Ussing-type chambers and bathed with Amphibian Ringer’s solution on both sides. Electrical potential difference, resistance, and short-circuit current (SCC) were measured by using calomel electrodes, Ag-AgCl electrodes, Ringer’s-agar bridges, and Tektronix digital multimeters. Under the conditions employed, SCC is a measure of net Na⫹ transport. The frog skin peptides bombesin, caerulein, and physalaemin were administered to the serosal side at concentrations of 0.5, 5.0, and 50 ng/ml. Control electrophysiological parameters were: potential difference, 23 ⫾ 2 mV; resistance, 738 ⫾ 59 ⍀ cm2; and SCC 32 ⫾ 3 A/cm2. Although bombesin and caerulein had no significant, reversible effect on potential difference, resistance, or SCC, physalaemin significantly, and reversibly, depressed SCC by 22%. Caerulein did significantly depress SCC, but the response was not reversible. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Amphibian; Rana pipiens; Bioactive peptides of the skin; Cutaneous Na⫹ transport
1. Introduction Frog skin produces a wide variety of polypeptides whose structure, but not function, in amphibians, is well known. There are several groups of these peptides. One, the tachykinins, is represented by a mammalian brain peptide known as substance-P. A frog-skin peptide that shows sequence homology with substance-P comes from the frog Physaleamis fusculamatus and is called physalaemin [5]. A second group is called the caeruleins. The first of these to be isolated was caerulein from Hyla caeruleia [2]. Caerulein is related to the mammalian hormone cholecystokinin-pancreozymin [4]. A third group is called the bombesin-like peptides. Bombesin was first isolated from the skin of Bombina bombina [1]. It was not until after the discovery of bombesin in amphibian skin that a mammalian counterpart— gastrin-releasing peptide—was discovered [4]. Although there are data describing actions of these peptides in mammalian systems, there is very little on their actions in frog skin. Transport of water and electrolytes are two of the important physiological functions of amphibian * Corresponding author. Tel.: ⫹1-909-869-4061; fax: ⫹1-909-8694078. E-mail address: [email protected] (D.F. Stiffler)
skin that might be modulated by one or more of the peptides. When placed on the outside (mucosal) surface of Rana pipiens skin, caerulein (pmolar concentrations) caused an increase in short-circuit current (SCC) that was interpreted to result from increased net Na⫹ transport across the skin [9]. Physalaemin had no effect on water permeability when used alone on the serosal side of the toad bladder; it was capable of abolishing the hydroosmotic responses to vasopressin and oxytocin [7]. Bombesin may stimulate Na⫹ transport in the mammalian kidney [6]. The objectives of the present study were to evaluate the serosal effects of bombesin, caerulein, and physalaemin on electrical potential difference (PD), SCC, and resistance (R) in the skin of the frog R. pipiens.
2. Materials and methods Frogs (R. pipiens) were purchased from commercial suppliers. The frogs were pithed, and a piece of ventral skin was mounted on one end of a glass cylinder. A plastic cap with an opening equal to the diameter of the opening in the glass cylinder (3.14 cm2) was constructed to fit over the piece of frog skin that was stretched over the end of the
0196-9781/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 9 9 ) 0 0 1 2 8 - X
1240
D.F. Stiffler / Peptides 20 (1999) 1239 –1241
Fig. 1. Frog-skin SCC responses to to serosal bombesin (open circles), caerulein (closed circles), and physalaemin (open triangles). The numbers of observations are in parentheses. * Physalaemin-treated skins were significantly different than controls at a dose of 50 ng/ml (P ⬍ 0.05; Tukey’s test for multiple comparisons). When the physalaemin was removed, the SCC significantly reversed to control levels (P ⬍ 0.05; Tukey’s test). The results of one-way analysis of varience were F(4,35) ⫽ 4.18; P ⫽ 0.0072. Bombesin-treated skins appeared to decrease SCC but the results were not significant [F(4,30) ⫽ 1.54]; P ⫽ 0.2166. Caerulein, like physalaemin, caused a significant decrease in SCC (*) at 50 ng/ml (P ⬍ 0.05; Tukey’s test) but unlike with physalaemin the decrease was not reversible. The results of one-way ANOVA were F(4,35) ⫽ 3.19; P ⫽ 0.0247.
glass cylinder (serosa in). An o-ring was placed inside the plastic cap to seal the skin tightly against the glass, and silicone grease was used on both sides of the skin to increase the seal. The plastic cap was held in place with rubber bands that attached to hooks on both the cylinder and cap. The cylinder with frog skin was filled with Ringer’s solution (composition: NaCl, 6.5 g/l; KCl, 0.14 g/l; CaCl2, 0.12 g/l; NaHCO3, 1.26 g/l). The Ringer’s filled cylinder apparatus then was suspended in Ringer’s solution so that there was no pressure gradient across the skin. PD-electrodes (Ringer’s-3% agar bridges) were attached to calomel electrodes that were attached to the input poles of a Tektronix DM 502 multimeter (Beaverton, OR, USA). SCC electrodes (saturated KCl-agar bridges) were placed with their ends opposite the center of the skin in both chambers. These current-passing bridges were connected to another DM 502 multimeter via Ag-AgCl electrodes. A 90-V dry cell battery and potentiometer were inserted in the circuit to null the spontaneous potential difference exhibited by the frog skin. The current required to accomplish this is the SSC and is equal to net Na⫹ flux under these conditions [11]. The ratio of the PD and SCC is the electrical resistance (⍀ cm2) that is directly related to ionic permeability. Once the skins were mounted, the potential difference was allowed to stabilize (1 h) before the experiments were started. The peptides were added to the Ringer’s solution in the cylinder (serosa) so that the final concentration in the cylinder equilibrated at 0.5, 5.0, and 50.0 ng/ml for approximately 20 min at each concentration. After the final concentration was tested, the chamber was flushed and refilled
with fresh Ringer’s solution. The peptides were purchased from Sigma (St. Louis, MO, USA).
3. Results and discussion The mean PD for the skins was 23 ⫾ 2 mV. The mean SCC was 32 ⫾ 3 A/cm2. The mean R was 738 ⍀ cm2. Of the three peptides, physalaemin had the greatest effect. Serosal treatment produced a decrease in SCC (Fig. 1), becoming significant (P ⬍ 0.05) at 50 ng/ml. Removal of the peptide resulted in a clear recovery (P ⬍ 0.05) as SCC (Fig. 1) returned to pre-control values. Caerulein, applied to the serosal surface, also produced a significant (P ⬍ 0.05) decrease in SCC; however, this decrease did not clearly reverse when the caerulein was removed. Although the recovery SCC was not significantly different than the 50 ng/ml treatment SCC, it was also not significantly different than the control SCC (0.05 ⬍ P ⬍ 0.1). Neither caerulein nor physalaemin caused significant changes in PD or R. Bombesin, applied to the serosal surface, had no significant effect on PD, SCC, or R. Physalaemin-like tachykinins have been detected in the blood of R. pipiens at 1.1–2 ng/g [3]. Circulating values for bombesin and caerulein have not been published; however, effective doses range from ng/kg to g/kg in a wide variety of systems [4]. The decrease in SCC induced by physalaemin and perhaps caerulein may have resulted from a decrease in active Na⫹ transport. Amphibian skin has been reported to contain highly polar substances that inhibit Na⫹, K⫹-ATPase [8]. Serosal applications of caerulein bring about different
D.F. Stiffler / Peptides 20 (1999) 1239 –1241
responses (inhibition of SCC) than do mucosal applications that stimulate SCC [9]. Caerulein interacts with dopaminergic neurons [4], which may explain this difference. Dopamine inhibits Na⫹ transport in frog kidney tubules [10].
References [1] Anastasi A, Erspamer V, Bucci M. Isolation and amino acid sequence of alytesin and bombesin, two analogous tetradecapeptides from the skin of European discoglossid frogs. Arch Biochem Biophys 1972; 148:443– 6. [2] Anastasi A, Erspamer V, Endean R. Isolation and amino acid sequence of caerulein, the active peptide in the skin of Hyla caerulea. Arch Biochem Biophys 1968;125:57– 68. [3] Creagh T, Skrabanek P, Cannon D, Balfe A, Powell D. Phylogeny of substance P. Gen Comp Endocrinol 1980;40:503– 6. [4] Erspamer V. Bioactive secretions of the integument. In: Heatwole H, Barthalmus GT, editors. Amphibian Biology, The Integument, Vol. 1. Chipping Norton, Australia: Surrey Beatty & Sons, 1994. pp. 179 –350.
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[5] Erspamer V, Anastasi A, Cei JM. Structure and pharmacological actions of physalaemin, the main active polypeptide of the skin of Physalaemis fuscumaculatus. Experientia 1964;20:489 –90. [6] Erspamer V, Melchiorri P, Sopranzi N. The action of bombesin on the kidney of the anesthetized dog. Br J Pharmacol 1973;48:438 – 55. [7] Furtado MFR. Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder. Effects of bradykinin, kallidin, eledoisin, and physalaemin. J Memb Biol 1971;4:165–78. [8] Flier J, Edwards MW, Daly JW. Widespread occurrance in frogs and toads of skin compounds interacting with ouabain sites and Na⫹, K⫹-ATPase. Science 1980;208:503–5. [9] Greenwell JR, Low HS. Action of caerulein, gastrin 17, pentagastrin, and secretin on active transport of sodium by frog Rana pipiens skin. J Memb Biol 1981;61:91– 6. [10] Hagiwara N, Kubota T, Kubakawa M, Fujimoto M. Effects of dopamine on the transport of Na, H, and Ca in the bullfrog kidney tubule. Jpn J Physiol 1990;40:351– 68. [11] Ussing HH, Zerahn K. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol Scand 1951;23:110 –27.