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CONTRACTILE EFFECTS OF PORCINE GALANIN(1–29)-NH2ON THE RAT ISOLATED GASTRIC FUNDUS: MEDIATION BY POTASSIUM IONS
Pharmacological Research, Vol. 36, No. 2, 1997
CONTRACTILE EFFECTS OF PORCINE GALANIN ( 1 – 29 ) -NH 2 ON THE RAT ISOLATED GASTRIC FUNDUS: MEDIATION BY POTASSIUM IONS R. KOROLKIEWICZU , K. TAKEUCHI†, W. SLIWINSKI‡, Z. KONSTANSKI, P. REKOWSKI§ and A. SZYK§ Department of Pharmacology, Medical Uni®ersity of Gdansk, Do Studzienki 38, 80-227 Gdansk, Poland, †Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical Uni®ersity, Misasagi, Yamashina, Kyoto 607, Japan, ‡2nd Department of Gynaecology and Obstetrics, Medical Uni®ersity of Gdansk, Kliniczna 1a, 80-402 Gdansk, Poland and §Faculty of Chemistry, Uni®ersity of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland Accepted 21 July 1997
Galanin Ž3]300 nM. evoked reproducible concentration-dependent contractions of rat isolated gastric fundus strips, EC 50 of the peptide equalled 16.7 nM Ž6.2]39.2. and the slope of the concentration]response curve was 34.8 Ž24.0]45.7.. The maximal response ŽE max . to carbachol Ž30 nM. was not affected by the absence of the potassium ions in the bathing solution. On the contrary the E max to galanin Ž300 nM. was decreased by almost 95% by the use of the potassium-free buffer. Re-exposure of the muscle strips to potassium containing bathing medium reversed the inhibition by about 35%, yet the value remained significantly lower than that of the control. Apamin Ž1 and 2 m M., glybenclamide Ž10 m M., clofilium tosylate Ž10 m M. did not significantly influence the E max to carbachol. Apamin or glybenclamide did not affect the contractile action of galanin, while clofilium attenuated the E max to the peptide in a concentration-dependent manner, the EC 50 of the agent being 9.44 m M Ž164 nM-541 m M.. It was concluded that the potassium ions play a modulatory role in gastric smooth muscle contraction following galanin receptor stimulation, probably by interacting with the extracellular calcium influx. Q 1997 The Italian Pharmacological Society KEY
WORDS:
galanin, potassium channel blockers, gastric smooth muscle.
INTRODUCTION Galanin is a 29-amino acid peptide isolated for the first time from the extracts of porcine ileum by Tatemoto et al. w1x. Galanin and galanin immunoreactivity are widely distributed in the central and peripheral nervous systems, gastrointestinal and urogenital tracts and the adrenal medulla of several mammalian species including man w2]5x. There is enough evidence to prove the existence of galanin binding sites in the intestinal, gastric smooth muscles and gastric mucosa w6]8x and it has been implicated in the physiological regulation of the smooth muscle motility, causing both contraction of some w7, 9x and relaxation of other smooth muscles w10x. Due to the lack of the specific antagonists relatively little U
Corresponding author. Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607, Japan.
1043]6618r97r080147]05r$25.00r0rfr970217
has been published on the mechanism of galanin action in the gastrointestinal smooth muscles w11x. The concentration-dependent block of galanin action by nifedypine and diltiazem imply an important role for the influx of extracellular Ca2q and a far lesser meaning for the release of Ca2q from the intracellular stores in the myotropic action of the peptide w12, 13x. Our study presents the results of the experiments designed to investigate the role of the extracellular potassium ions in the contractile action of galanin on rat gastric fundus strips.
MATERIALS AND METHODS
Animals and tissue preparation Albino]Wistar rats of either gender Žweighing 180]250 g. were housed in mesh-wire bottom cages and kept in standard laboratory conditions Ža natural light]dark cycle., with food ŽMurigran chow pelQ1997 The Italian Pharmacological Society
148
lets, Bacutil, Motycz, Poland. and tap water ad libitum. Rats were fasted overnight before the experiments. Animals were killed by cervical dislocation. The abdomen was opened with a midline incision, the stomach excised, the fundus dissected out and longitudinal muscle strips were prepared according to Vane w14x. Strips were placed in Tyrode’s solution Žfor composition see below. at 48C and bubbled with O 2 ]CO 2 Ž95:5.. Three to 5 min later the strips were suspended vertically in water-jacketed, siliconized organ baths Žvolume 15 ml. containing Tyrode’s solution, with the thermostat set at 378C, aerated continuously with O 2 ]CO 2 Ž95:5. and kept at a resting tension of 2.0 g. The responses of the stomach strips were recorded isotonically. One end of the strip was attached to a fixed support and the free one to a lever connected via a spring to PIT 212 force displacement transducers ŽCOTM, Białystok, Poland.. Transducers were joined to a TZ 4100 line recorder ŽLaboratorni Pristroje, Prague, Czech Republic.. Composition of the Tyrode buffer ŽpH 7.4. was as follows Žin mM.: NaCl 136.9; KCl 3.35; CaCl 2 1.46; MgCl 2 1.03; NaHCO3 11.9; NaH 2 PO4 0.48; glucose 5.0. Potassium-free Tyrode contained Žin mM.: NaCl 140.25; CaCl 2 1.46; MgCl 2 1.03; NaHCO3 11.9; NaH 2 PO4 0.48; glucose 5.0. In order to eliminate the probable involvement of cholinergic and adrenergic components in the investigated responses, the studies were carried out in the presence of atropine sulphate Ž1 m M. and guanethidine Ž3 m M.. To avoid the proteolytic degradation of galanin the aminopeptidase and endopeptidase inhibitors were included in the incubation buffer: amastatin Ž10 m M. and phosphoramidon Ž1 m M.. In our previous experiments we determined that these inhibitors at the concentration used did not affect the contraction of rat gastric fundus strips. Tissues were allowed to equilibrate for 60 min before the beginning of the experiment. The buffer was changed every 5 min, except for the contact time of the test agent with the tissue, lasting 15]30 min.
CONCENTRATION–CONTRACTION CURVES FOR PORCINE GALANIN The experiments were started when reproducible contractile responses to carbamylcholine chloridecarbachol Ž10]30 nM. were obtained. No more than two concentration]contraction studies have been performed on each strip. Galanin was added in increasing concentrations Žhalf-logarithmic increments. directly into the organ baths by cumulative addition}each concentration added when the effect of the preceding one had reached a steady state, until maximum contraction occurred Ži.e. when the contractile response could not be increased further by a higher concentration of peptide.. In our previous reports w9, 12, 15x we have repeatedly shown that cumulative additions of galanin do not seem to
Pharmacological Research, Vol. 36, No. 2, 1997
evoke tachyphylaxis in isolated gastric fundus strips, as we were able to repeat full concentration]contraction procedures for galanin at 30-min intervals and still produce consistent responses. The contact time of the peptide with the muscle strip allowed for the development of maximum contraction ranged from 15 to 20 min. Three minutes after the maximum contraction was reached the tissue was washed out at a rate of 1.2 ml sy1 for 2]3 min until the length of the strip returned to the basal level. The strip was then left to equilibrate for 30 min, the buffer was changed every 5 min. The viability and reproducible contractility of each strip was examined at the end of each experimental session by a submaximal contractile response to carbachol at the same concentrations as at the start. INFLUENCE OF POTASSIUM-FREE BUFFER AND POTASSIUM-CHANNEL BLOCKERS ON THE MYOTROPIC ACTIONS OF GALANIN Experiments were conducted in the same set as described above. When examining the influence of potassium-free Tyrode buffer on the contractile action of galanin and carbachol; muscle strips were exposed to 300 nM galanin Žconcentration established to have caused the maximal contraction. or 30 nM carbachol in the organ baths containing standard, potassium-containing Tyrode’s solution Žcontrol.. When the maximum contraction developed the tissues were washed out until the length of the strips returned to the basal level. Immediately the buffer was changed to potassium-free Tyrode’s solution and a 30-min equilibration period ensued, buffer in the organ baths was changed every 5 min. Then 300 nM of galanin or 30 nM of carbachol were added into the organ baths. Three minutes after the development of maximum contractions the tissues were washed out until the length of the strips returned to the basal level. Again the buffer was changed to the standard, potassium-containing Tyrode’s solution and after another 30 min of equilibration the strips were exposed either to 300 nM of galanin or 30 nM of carabachol. In the case of the experiments involving potassium channel blockers no more than two contraction studies were performed on each strip, one in the absence Žcontrol. and the other in the presence of increasing concentrations of glybenclamide, apamin, clofilium tosylate, applied alternately. Galanin was added to the bath at a concentration of 300 nM. Three minutes after the maximum contraction was reached the tissue was washed out until the length of the strip returned to the basal level. The time gap between control and exposure to a test agent was a 30-min equilibration period, the buffer was changed every 5 min. The contact times of all the test agents with the muscle strips were 15]20 min. Carbachol and clofilium tosy-
Pharmacological Research, Vol. 36, No. 2, 1997
late were dissolved in Tyrode solution at the roomtemperature. Glybenclamide was solubilized in 1 ml of dimethyl sulfoxide ŽDMSO., apamin in 1 ml of 0.05 M acetic acid, respectively, and adjusted to the desired volume with Tyrode’s buffer. In experiments with glybenclamide and apamin the effects of DMSO and 0.05 M acetic acid were studied on muscle contractility to galanin. These results are presented as a percentage of maximal responses to galanin with DMSO or galanin with acetic acid in the concentrations used for dissolving glybenclamide and apamin.
DRUGS NaCl, KCl, CaCl 2 , MgCl 2 , NaH 2 PO4, glucose and 2 acetic acid solution were purchased from P.P.H. ŽPolskie Odczynniki Chemiczne, Gliwice, Poland., carbachol, apamin, DMSO, atropine sulphate, guanethidine, amastatin and phosphoramidon from Sigma ŽSt Louis, MO, USA., glybenclamide from Polpharma ŽStarogardzkie Zaklady Farmaceutyczne, Starogard Gdanski, Poland., clofilium tosylate from RBI ŽNatick, MA, USA.. Porcine galanin was synthesized by Rekowski and Szyk using a solid phase peptide synthesis and purified to homogeneity by high performance liquid chromatography ŽHPLC, system Gold Beckman Chromatograph; for details see w9x.. M
BIOSTATISTICAL ANALYSIS OF DATA Results were expressed as a percentage of the maximum response induced by galanin or carbachol. Maximum reponses ŽE max s. produced by galanin or carbachol either in the potassium-free medium or in the presence of the test agents were expressed as a percentage in comparison to the respective controls, i.e. values of contraction evoked by 300 nM galanin
149
or 30 nM carbachol in standard, potassium-containing Tyrode’s solution or in the absence of the pharmacological modulator. EC 50 values of galanin and clofilium and the slope of the galanin’s concentration]contraction curve were estimated using Pharmacological Calculation System-PharmrPCS, version 4 computer program based on the Manual of Pharmacologic Calculations with Computer Programs w16x. E max values, EC 50 values and the slope of the galanin concentration]contraction curve are expressed as means with 95% confidence limits. E max values were compared to controls using One-Way Analysis of Variance ŽANOVA. plus Bonferroni post-ANOVA test. To perform ANOVA plus the Bonferroni post-ANOVA test a GraphPAD INSTAT computer programme version 1.12a was used. The two-tailed P- 0.05 values were taken to indicate a significant difference.
RESULTS
Effects of galanin on rat gastric fundus strips Galanin evoked concentration-dependent contractions of rat gastric fundus strips, giving reproducible results at 3 nM and a maximum response at 300 nM, yielding a typically shaped concentration]contraction curve with EC 50 of 16.7 nM Ž6.2]39.2., the slope 34.8 Ž24.0]45.7. and a fall-down effect at the supramaximal concentrations Ždata not shown..
Effects of potassium-free buffer on the action of carbachol and galanin The E max to carbachol was not affected by the absence of potassium in Tyrode’s solution, the contraction amounting to 101% of control. However, the E max of galanin was significantly decreased by almost 95% by the use of the potassium-free buffer ŽTable I.. Re-exposure of the muscle strips to potassium-containing Tyrode’s solution for 30 min considerably reversed the above mentioned inhibition
Table I Contractile effects of carbachol and galanin on rat isolated gastric fundus strips obtained in the absence and presence of potassium in the incubation buffer Contractile agents
Concentration (nM)
Contraction (%) in potassium-free bathing medium
Contraction (%) in potassium-containing bathing medium
Carbachol
30
101 Ž73.3]151. UUU 5.53 Ž3.6]6.7.
97.9 Ž91.5]123. UUU 39.9 ,††† Ž24.5]57.3.
Galanin
300
The results are expressed as means with the 95% confidence limits. The upper and lower confidence limits are given in parentheses. E max values produced by carbachol or galanin in potassium-free Tyrode’s solution are expressed as a percentage in comparison to the respective controls Žthe action of carbachol and galanin in standard Tyrode’s solution. and UUU P - 0.001 contraction in potassium-free medium were compared using ANOVA plus Bonferroni post-ANOVA test. compared with contraction in the potassium-containing Tyrode’s solution Žcontrol.. †††P- 0.001 contraction in the potassium-containing Tyrode’s solution Žre-exposure. compared with potassium-free bathing medium.
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by about 35%. Nevertheless, the value remained remarkably lower than that of the control ŽTable I..
The action of some potassium channel blockers upon the contraction e®oked by carbachol and galanin DMSO and acetic acid in the concentrations used did not significantly modify E max values to galanin or carbachol Žvalues not shown.. Apamin 1 and 2 m M, glybenclamide up to 10 m M did not change the E max values to carbachol and galanin. Clofilium tosylate up to 10 m M did not markedly influence the E max to carbachol ŽTable II., however, the agent attenuated the contraction evoked by galanin in a concentration-dependent manner, its EC 50 being 9.44 m M Ž164 nM ]541 m M..
DISCUSSION Grider and Makhlouf w17x were able to demonstrate the role of potassium in the mediation of galanin’s action in the isolated guinea-pig and human gastric smooth muscle cells only in the presence of VIP acting as a smooth muscle relaxant via the cyclic adenosine monophospate mechanism. We have clearly shown that the myotropic effects of galanin on the gastric fundus strips were strongly suppressed in the potassium-free environment ŽTable I. and in the concentration-dependent manner by the potassium channel blocker clofilium tosylate. Taking into account that potassium ion-containing salt, i.e. KCl was able to induce contraction through membrane depolarization by increasing the permeability of the cell membrane to Ca2q w18x and that galanin evoked
contraction of gastrointestinal smooth muscle cells depend to a great extent on the influx of the extracellular Ca2q w12, 13x it was not surprising that the absence of potassium ions in the bathing solution or the use of clofilium decreased the effect of galanin. On the other hand, it is widely known that the activity of calcium and potassium channels can be modulated by excitatory and inhibitory neurotransmitters w19x. The inability of potassium-containing Tyrode’s solution to fully reverse the inhibitory effect of the potassium ion lack when the excitatory effects of galanin are concerned might suggest that the potassium deficiency might have caused some functional changes in gastric smooth muscle cells affecting the contractile potency of galanin. However, the exact molecular mechanism cannot be determined on the basis of our experiments alone. We showed that glybenclamide an ATP-sensitive Kq channel antagonist w20x in concentrations up to 10 m M and apamin Ž1 or 2 m M., a calcium-activated potassium channel blocker w21x did not affect the action of galanin on rat gastric fundus. The decrease in the contractile force of galanin seems to be specific as the responses to the added carbachol were unaffected by potassium-free medium or any of the potassium channel blockers used, at least over a relatively short time span. The results of the protocols employing potassium-free Tyrode solution and the use of clofilium provide complementary data consistent with potassium ions playing a modulatory role in gastric smooth muscle contraction following galanin receptor stimulation. It does not seem unreasonable to speculate that the potassium ions interact and influence the extracellular calcium currents after binding of galanin to its gastric fundus
Table II Contractile actions of galanin as obtained in the presence of some potassium channel blockers Potassium channel blockers
Concentration (m M.
Contractile agents
Concentration (n M.
Contractions in the presence of the potassium channel blocker (%)
10
Galanin
300
Apamin
1
Galanin
300
Apamin
2
Galanin
300
10
Carbachol
30
2
Carbachol
30
10
Carbachol
30
91.5 Ž64.3]107. 88.2 Ž67.8]132. 82.5 Ž75.9]88.5. 99.7 Ž80.0]109. 95.2 Ž78.1]112. 101 Ž91.6]111.
Glybenclamide
Glybenclamide Apamin Clofilium tosylate
The contractile responses produced by the tested agents were measured before Žcontrol. and after the use of a particular potassium channel blocker. In experiments with glybenclamide and apamin the results are presented as a percentage of maximal responses to galanin with DMSO or galanin with 0.05 M acetic acid the concentration used for dissolving glybenclamide and apamin, respectively. The incubation time of the antagonists with the tissues was 15]20 min. The results were calculated as means of the respective controls with 95% confidence limits. They were compared using ANOVA plus Bonferroni post-ANOVA test.
Pharmacological Research, Vol. 36, No. 2, 1997
receptor. Although the present results do not allow us to pinpoint particular ionic channels involved in the process as clofilium tosylate at similar concentration to that used in the study tends to affect multiple inward and outward currents in guinea pig ventricular myocytes w22x, they warrant further investigations into the area.
ACKNOWLEDGEMENTS R.K. is a Japan Society for Promotion of Science fellow and a recipient of Foundation for Science Reward and is on sabbatical from the Department of Pharmacology, Medical University of Gdansk, Poland. The cost of galanin synthesis was deferred from the Polish State Committee for Scientific Research ŽKBN. grant number BW-8000-5-0190-7.
REFERENCES 1. Tatemoto K, Rıkaeus A, Jornvall H, McDonald TJ, ˆ ¨ Mutt V. Galanin-a novel biologically active peptide from porcine intesting. FEBS lett 1983; 164: 124]8. 2. Amiranoff B, Servin Al, Rouyer-Fessard C, Couvineau A, Tatemoto K, Laburthe M. Galanin receptors in a hamster pancreatic b-cell tumor: identification and molecular characterization. Endocrinology 1987; 121: 284]9. 3. Bartfai T. Galanin: a neuropeptide with important central nervous system action. In: Bloom FE, Kupfer DJ, eds. Psychopharmacology: the Fourth Generation of Progress. New York: Raven Press Ltd, 1995:563]71. 4. Melander T, Hokfelt T, Rokaeus A, Fahrenkrug J, ¨ ¨ Tatemoto K, Mutt V. Distribution of galanin-like immunoreactivity in the gastro-intestinal tract of several mammalian species. Cell Tiss Res 1985; 239: 253]70. 5. Rokaeus A. Galanin: a newly isolated biologically ¨ active neuropeptide. Trends Neurosci 1987; 10: 158]64. 6. Rossowski WJ, Rossowski TM, Zacharia S, Ertan A, Coy DH. Galanin binding sites in rat gastric and jejunal smooth muscle membranes preparations. Peptides 1990; 11: 333]8. 7. Katsoulis S, Schmidt WE, Schworer ¨ H, Creutzfeldt W. Effects of galanin, its analogues and fragments on rat isolated fundus strips. Br J Pharmacol 1990; 101: 297]300. 8. Lorimer DD, Benya RV. Cloning and quantification of galanin-1 receptor expression by mucosal cells
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lining the human gastrointestinal tract. Biochem Biophys Res Commun 1996; 22: 379]85. Korolkiewicz R. Sliwinski W, Rekowski P, HalamaBorowiec A, Mucha P, Szczurowicz A, Korolkiewicz KZ. Galanin, galantide and galanin Ž1-14.-w aaminobutyric acid 8 x-scyliorhinin-I: structure dependent effects on the rat isolated gastric fundus. Pharmacol Res 1997; 35: 7]16. Fox JET, McDonald TJ, Kostolanska F, Tatemoto K. Galanin: an inhibitory neural peptide in the canine samll intestine. Life Sci 1986; 39: 103]10. Gu ZFA, Rossowski WJ, Coy DH, Pradhan TK, Jensen RT. Chimeric galanin analogs that function as antagonists in the CNS are full agonist in gastrointestinal smooth muscle. J Pharmacol Exp Ther 1993; 266: 912]18. Sliwinski W, Korolkiewicz R, Rekowski P, Halama A, Korolkiewicz KZ. Actions of galanin and some of its analogues on rat isolated gastric fundus. Fundam Clin Pharmacol 1996; 10: 442]9. Fontaine J, Lebrun P. Galanin: Ca2q-dependent contractile effects on the isolated mouse distal colon. Eur J Pharmacol 1989; 164: 583]6. Vane JR. A sensitive method for the assay of 5-hydroxytryptamine. Br J Pharmacol 1957; 12: 344]9. Korolkiewicz R, Sliwinski W, Rekowski P, Halama A, Mucha P, Szczurowicz A, Guzowski P and Korolkiewicz KZ. Contractile action of galanin analogues on rat isolated fundus strips is modified by tachyphylaxis to substance P. Pharmacol Res 1996; 33: 361]5. Tallarida RJ, Murray RB. Manual of Pharmacologic calculations with computer programs. 2nd ed. New York: Springer Verlag, 1986. Grider JR, Makhlouf GM. The modulatory function of galanin: potentiation of VIP-induced relaxation in isolated smooth muscle cells. Gastroenterology 1988; 94: A157. Bolton TB. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol Re® 1979; 59: 606]718. Makhlouf GM. Neuromuscular function of the small intestine. In: LR Johnson, ed. Physiology of the Gastrointestinal Tract. New York: Raven Press Ltd., 1994:977]990. Jonas P, Koh D-S, Kampe K, Hermsteiner M, Vogel V. ATP-sensitive Ca-activated K channels in vertebrate axons: novel links between metabolism and excitability. Pflugers Arch 1991; 418: 68]73. ¨ Aronson JK. Potassium channels in nervous tissue. Biochem Pharmacol 1992; 43: 11]14. Li Q, Himmel HM, Ravens U. Selectivty of class-III antiarrhytmic action of clofilium in guinea pig ventricular myocytes. J Cardio®asc Pharmacol 1996; 27: 401]10.
CONTRACTILE EFFECTS OF PORCINE GALANIN ( 1 – 29 ) -NH 2 ON THE RAT ISOLATED GASTRIC FUNDUS: MEDIATION BY POTASSIUM IONS R. KOROLKIEWICZU , K. TAKEUCHI†, W. SLIWINSKI‡, Z. KONSTANSKI, P. REKOWSKI§ and A. SZYK§ Department of Pharmacology, Medical Uni®ersity of Gdansk, Do Studzienki 38, 80-227 Gdansk, Poland, †Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical Uni®ersity, Misasagi, Yamashina, Kyoto 607, Japan, ‡2nd Department of Gynaecology and Obstetrics, Medical Uni®ersity of Gdansk, Kliniczna 1a, 80-402 Gdansk, Poland and §Faculty of Chemistry, Uni®ersity of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland Accepted 21 July 1997
Galanin Ž3]300 nM. evoked reproducible concentration-dependent contractions of rat isolated gastric fundus strips, EC 50 of the peptide equalled 16.7 nM Ž6.2]39.2. and the slope of the concentration]response curve was 34.8 Ž24.0]45.7.. The maximal response ŽE max . to carbachol Ž30 nM. was not affected by the absence of the potassium ions in the bathing solution. On the contrary the E max to galanin Ž300 nM. was decreased by almost 95% by the use of the potassium-free buffer. Re-exposure of the muscle strips to potassium containing bathing medium reversed the inhibition by about 35%, yet the value remained significantly lower than that of the control. Apamin Ž1 and 2 m M., glybenclamide Ž10 m M., clofilium tosylate Ž10 m M. did not significantly influence the E max to carbachol. Apamin or glybenclamide did not affect the contractile action of galanin, while clofilium attenuated the E max to the peptide in a concentration-dependent manner, the EC 50 of the agent being 9.44 m M Ž164 nM-541 m M.. It was concluded that the potassium ions play a modulatory role in gastric smooth muscle contraction following galanin receptor stimulation, probably by interacting with the extracellular calcium influx. Q 1997 The Italian Pharmacological Society KEY
WORDS:
galanin, potassium channel blockers, gastric smooth muscle.
INTRODUCTION Galanin is a 29-amino acid peptide isolated for the first time from the extracts of porcine ileum by Tatemoto et al. w1x. Galanin and galanin immunoreactivity are widely distributed in the central and peripheral nervous systems, gastrointestinal and urogenital tracts and the adrenal medulla of several mammalian species including man w2]5x. There is enough evidence to prove the existence of galanin binding sites in the intestinal, gastric smooth muscles and gastric mucosa w6]8x and it has been implicated in the physiological regulation of the smooth muscle motility, causing both contraction of some w7, 9x and relaxation of other smooth muscles w10x. Due to the lack of the specific antagonists relatively little U
Corresponding author. Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607, Japan.
1043]6618r97r080147]05r$25.00r0rfr970217
has been published on the mechanism of galanin action in the gastrointestinal smooth muscles w11x. The concentration-dependent block of galanin action by nifedypine and diltiazem imply an important role for the influx of extracellular Ca2q and a far lesser meaning for the release of Ca2q from the intracellular stores in the myotropic action of the peptide w12, 13x. Our study presents the results of the experiments designed to investigate the role of the extracellular potassium ions in the contractile action of galanin on rat gastric fundus strips.
MATERIALS AND METHODS
Animals and tissue preparation Albino]Wistar rats of either gender Žweighing 180]250 g. were housed in mesh-wire bottom cages and kept in standard laboratory conditions Ža natural light]dark cycle., with food ŽMurigran chow pelQ1997 The Italian Pharmacological Society
148
lets, Bacutil, Motycz, Poland. and tap water ad libitum. Rats were fasted overnight before the experiments. Animals were killed by cervical dislocation. The abdomen was opened with a midline incision, the stomach excised, the fundus dissected out and longitudinal muscle strips were prepared according to Vane w14x. Strips were placed in Tyrode’s solution Žfor composition see below. at 48C and bubbled with O 2 ]CO 2 Ž95:5.. Three to 5 min later the strips were suspended vertically in water-jacketed, siliconized organ baths Žvolume 15 ml. containing Tyrode’s solution, with the thermostat set at 378C, aerated continuously with O 2 ]CO 2 Ž95:5. and kept at a resting tension of 2.0 g. The responses of the stomach strips were recorded isotonically. One end of the strip was attached to a fixed support and the free one to a lever connected via a spring to PIT 212 force displacement transducers ŽCOTM, Białystok, Poland.. Transducers were joined to a TZ 4100 line recorder ŽLaboratorni Pristroje, Prague, Czech Republic.. Composition of the Tyrode buffer ŽpH 7.4. was as follows Žin mM.: NaCl 136.9; KCl 3.35; CaCl 2 1.46; MgCl 2 1.03; NaHCO3 11.9; NaH 2 PO4 0.48; glucose 5.0. Potassium-free Tyrode contained Žin mM.: NaCl 140.25; CaCl 2 1.46; MgCl 2 1.03; NaHCO3 11.9; NaH 2 PO4 0.48; glucose 5.0. In order to eliminate the probable involvement of cholinergic and adrenergic components in the investigated responses, the studies were carried out in the presence of atropine sulphate Ž1 m M. and guanethidine Ž3 m M.. To avoid the proteolytic degradation of galanin the aminopeptidase and endopeptidase inhibitors were included in the incubation buffer: amastatin Ž10 m M. and phosphoramidon Ž1 m M.. In our previous experiments we determined that these inhibitors at the concentration used did not affect the contraction of rat gastric fundus strips. Tissues were allowed to equilibrate for 60 min before the beginning of the experiment. The buffer was changed every 5 min, except for the contact time of the test agent with the tissue, lasting 15]30 min.
CONCENTRATION–CONTRACTION CURVES FOR PORCINE GALANIN The experiments were started when reproducible contractile responses to carbamylcholine chloridecarbachol Ž10]30 nM. were obtained. No more than two concentration]contraction studies have been performed on each strip. Galanin was added in increasing concentrations Žhalf-logarithmic increments. directly into the organ baths by cumulative addition}each concentration added when the effect of the preceding one had reached a steady state, until maximum contraction occurred Ži.e. when the contractile response could not be increased further by a higher concentration of peptide.. In our previous reports w9, 12, 15x we have repeatedly shown that cumulative additions of galanin do not seem to
Pharmacological Research, Vol. 36, No. 2, 1997
evoke tachyphylaxis in isolated gastric fundus strips, as we were able to repeat full concentration]contraction procedures for galanin at 30-min intervals and still produce consistent responses. The contact time of the peptide with the muscle strip allowed for the development of maximum contraction ranged from 15 to 20 min. Three minutes after the maximum contraction was reached the tissue was washed out at a rate of 1.2 ml sy1 for 2]3 min until the length of the strip returned to the basal level. The strip was then left to equilibrate for 30 min, the buffer was changed every 5 min. The viability and reproducible contractility of each strip was examined at the end of each experimental session by a submaximal contractile response to carbachol at the same concentrations as at the start. INFLUENCE OF POTASSIUM-FREE BUFFER AND POTASSIUM-CHANNEL BLOCKERS ON THE MYOTROPIC ACTIONS OF GALANIN Experiments were conducted in the same set as described above. When examining the influence of potassium-free Tyrode buffer on the contractile action of galanin and carbachol; muscle strips were exposed to 300 nM galanin Žconcentration established to have caused the maximal contraction. or 30 nM carbachol in the organ baths containing standard, potassium-containing Tyrode’s solution Žcontrol.. When the maximum contraction developed the tissues were washed out until the length of the strips returned to the basal level. Immediately the buffer was changed to potassium-free Tyrode’s solution and a 30-min equilibration period ensued, buffer in the organ baths was changed every 5 min. Then 300 nM of galanin or 30 nM of carbachol were added into the organ baths. Three minutes after the development of maximum contractions the tissues were washed out until the length of the strips returned to the basal level. Again the buffer was changed to the standard, potassium-containing Tyrode’s solution and after another 30 min of equilibration the strips were exposed either to 300 nM of galanin or 30 nM of carabachol. In the case of the experiments involving potassium channel blockers no more than two contraction studies were performed on each strip, one in the absence Žcontrol. and the other in the presence of increasing concentrations of glybenclamide, apamin, clofilium tosylate, applied alternately. Galanin was added to the bath at a concentration of 300 nM. Three minutes after the maximum contraction was reached the tissue was washed out until the length of the strip returned to the basal level. The time gap between control and exposure to a test agent was a 30-min equilibration period, the buffer was changed every 5 min. The contact times of all the test agents with the muscle strips were 15]20 min. Carbachol and clofilium tosy-
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late were dissolved in Tyrode solution at the roomtemperature. Glybenclamide was solubilized in 1 ml of dimethyl sulfoxide ŽDMSO., apamin in 1 ml of 0.05 M acetic acid, respectively, and adjusted to the desired volume with Tyrode’s buffer. In experiments with glybenclamide and apamin the effects of DMSO and 0.05 M acetic acid were studied on muscle contractility to galanin. These results are presented as a percentage of maximal responses to galanin with DMSO or galanin with acetic acid in the concentrations used for dissolving glybenclamide and apamin.
DRUGS NaCl, KCl, CaCl 2 , MgCl 2 , NaH 2 PO4, glucose and 2 acetic acid solution were purchased from P.P.H. ŽPolskie Odczynniki Chemiczne, Gliwice, Poland., carbachol, apamin, DMSO, atropine sulphate, guanethidine, amastatin and phosphoramidon from Sigma ŽSt Louis, MO, USA., glybenclamide from Polpharma ŽStarogardzkie Zaklady Farmaceutyczne, Starogard Gdanski, Poland., clofilium tosylate from RBI ŽNatick, MA, USA.. Porcine galanin was synthesized by Rekowski and Szyk using a solid phase peptide synthesis and purified to homogeneity by high performance liquid chromatography ŽHPLC, system Gold Beckman Chromatograph; for details see w9x.. M
BIOSTATISTICAL ANALYSIS OF DATA Results were expressed as a percentage of the maximum response induced by galanin or carbachol. Maximum reponses ŽE max s. produced by galanin or carbachol either in the potassium-free medium or in the presence of the test agents were expressed as a percentage in comparison to the respective controls, i.e. values of contraction evoked by 300 nM galanin
149
or 30 nM carbachol in standard, potassium-containing Tyrode’s solution or in the absence of the pharmacological modulator. EC 50 values of galanin and clofilium and the slope of the galanin’s concentration]contraction curve were estimated using Pharmacological Calculation System-PharmrPCS, version 4 computer program based on the Manual of Pharmacologic Calculations with Computer Programs w16x. E max values, EC 50 values and the slope of the galanin concentration]contraction curve are expressed as means with 95% confidence limits. E max values were compared to controls using One-Way Analysis of Variance ŽANOVA. plus Bonferroni post-ANOVA test. To perform ANOVA plus the Bonferroni post-ANOVA test a GraphPAD INSTAT computer programme version 1.12a was used. The two-tailed P- 0.05 values were taken to indicate a significant difference.
RESULTS
Effects of galanin on rat gastric fundus strips Galanin evoked concentration-dependent contractions of rat gastric fundus strips, giving reproducible results at 3 nM and a maximum response at 300 nM, yielding a typically shaped concentration]contraction curve with EC 50 of 16.7 nM Ž6.2]39.2., the slope 34.8 Ž24.0]45.7. and a fall-down effect at the supramaximal concentrations Ždata not shown..
Effects of potassium-free buffer on the action of carbachol and galanin The E max to carbachol was not affected by the absence of potassium in Tyrode’s solution, the contraction amounting to 101% of control. However, the E max of galanin was significantly decreased by almost 95% by the use of the potassium-free buffer ŽTable I.. Re-exposure of the muscle strips to potassium-containing Tyrode’s solution for 30 min considerably reversed the above mentioned inhibition
Table I Contractile effects of carbachol and galanin on rat isolated gastric fundus strips obtained in the absence and presence of potassium in the incubation buffer Contractile agents
Concentration (nM)
Contraction (%) in potassium-free bathing medium
Contraction (%) in potassium-containing bathing medium
Carbachol
30
101 Ž73.3]151. UUU 5.53 Ž3.6]6.7.
97.9 Ž91.5]123. UUU 39.9 ,††† Ž24.5]57.3.
Galanin
300
The results are expressed as means with the 95% confidence limits. The upper and lower confidence limits are given in parentheses. E max values produced by carbachol or galanin in potassium-free Tyrode’s solution are expressed as a percentage in comparison to the respective controls Žthe action of carbachol and galanin in standard Tyrode’s solution. and UUU P - 0.001 contraction in potassium-free medium were compared using ANOVA plus Bonferroni post-ANOVA test. compared with contraction in the potassium-containing Tyrode’s solution Žcontrol.. †††P- 0.001 contraction in the potassium-containing Tyrode’s solution Žre-exposure. compared with potassium-free bathing medium.
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by about 35%. Nevertheless, the value remained remarkably lower than that of the control ŽTable I..
The action of some potassium channel blockers upon the contraction e®oked by carbachol and galanin DMSO and acetic acid in the concentrations used did not significantly modify E max values to galanin or carbachol Žvalues not shown.. Apamin 1 and 2 m M, glybenclamide up to 10 m M did not change the E max values to carbachol and galanin. Clofilium tosylate up to 10 m M did not markedly influence the E max to carbachol ŽTable II., however, the agent attenuated the contraction evoked by galanin in a concentration-dependent manner, its EC 50 being 9.44 m M Ž164 nM ]541 m M..
DISCUSSION Grider and Makhlouf w17x were able to demonstrate the role of potassium in the mediation of galanin’s action in the isolated guinea-pig and human gastric smooth muscle cells only in the presence of VIP acting as a smooth muscle relaxant via the cyclic adenosine monophospate mechanism. We have clearly shown that the myotropic effects of galanin on the gastric fundus strips were strongly suppressed in the potassium-free environment ŽTable I. and in the concentration-dependent manner by the potassium channel blocker clofilium tosylate. Taking into account that potassium ion-containing salt, i.e. KCl was able to induce contraction through membrane depolarization by increasing the permeability of the cell membrane to Ca2q w18x and that galanin evoked
contraction of gastrointestinal smooth muscle cells depend to a great extent on the influx of the extracellular Ca2q w12, 13x it was not surprising that the absence of potassium ions in the bathing solution or the use of clofilium decreased the effect of galanin. On the other hand, it is widely known that the activity of calcium and potassium channels can be modulated by excitatory and inhibitory neurotransmitters w19x. The inability of potassium-containing Tyrode’s solution to fully reverse the inhibitory effect of the potassium ion lack when the excitatory effects of galanin are concerned might suggest that the potassium deficiency might have caused some functional changes in gastric smooth muscle cells affecting the contractile potency of galanin. However, the exact molecular mechanism cannot be determined on the basis of our experiments alone. We showed that glybenclamide an ATP-sensitive Kq channel antagonist w20x in concentrations up to 10 m M and apamin Ž1 or 2 m M., a calcium-activated potassium channel blocker w21x did not affect the action of galanin on rat gastric fundus. The decrease in the contractile force of galanin seems to be specific as the responses to the added carbachol were unaffected by potassium-free medium or any of the potassium channel blockers used, at least over a relatively short time span. The results of the protocols employing potassium-free Tyrode solution and the use of clofilium provide complementary data consistent with potassium ions playing a modulatory role in gastric smooth muscle contraction following galanin receptor stimulation. It does not seem unreasonable to speculate that the potassium ions interact and influence the extracellular calcium currents after binding of galanin to its gastric fundus
Table II Contractile actions of galanin as obtained in the presence of some potassium channel blockers Potassium channel blockers
Concentration (m M.
Contractile agents
Concentration (n M.
Contractions in the presence of the potassium channel blocker (%)
10
Galanin
300
Apamin
1
Galanin
300
Apamin
2
Galanin
300
10
Carbachol
30
2
Carbachol
30
10
Carbachol
30
91.5 Ž64.3]107. 88.2 Ž67.8]132. 82.5 Ž75.9]88.5. 99.7 Ž80.0]109. 95.2 Ž78.1]112. 101 Ž91.6]111.
Glybenclamide
Glybenclamide Apamin Clofilium tosylate
The contractile responses produced by the tested agents were measured before Žcontrol. and after the use of a particular potassium channel blocker. In experiments with glybenclamide and apamin the results are presented as a percentage of maximal responses to galanin with DMSO or galanin with 0.05 M acetic acid the concentration used for dissolving glybenclamide and apamin, respectively. The incubation time of the antagonists with the tissues was 15]20 min. The results were calculated as means of the respective controls with 95% confidence limits. They were compared using ANOVA plus Bonferroni post-ANOVA test.
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receptor. Although the present results do not allow us to pinpoint particular ionic channels involved in the process as clofilium tosylate at similar concentration to that used in the study tends to affect multiple inward and outward currents in guinea pig ventricular myocytes w22x, they warrant further investigations into the area.
ACKNOWLEDGEMENTS R.K. is a Japan Society for Promotion of Science fellow and a recipient of Foundation for Science Reward and is on sabbatical from the Department of Pharmacology, Medical University of Gdansk, Poland. The cost of galanin synthesis was deferred from the Polish State Committee for Scientific Research ŽKBN. grant number BW-8000-5-0190-7.
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