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FMRF-NH2-induced contraction might be resistant to organic Ca2+-antagonists in molluscan smooth muscle
2417
agonists induced contractions only in the presence of extracellular Ca, and carbachol desensitized McN-A-343- or AHR-602-induced contraction. Desensitization by carbachol was also observed in K-induced contraction, believed to occur by Ca influx through the voltage-dependent Ca channels activated by X. It is possible that ca~bachol desensitizes the McN-A-343-, AHR-602- or K-induced contraction by suppressing the function of Ca channels in the plasma membrane. However, it is also possible that contraction due to McN-A-343, AHR-602 or K is influenceP by changes in intracellular Ca stores. Involvement of intracellular Ca stores in the contraction elicited influx of extracellular Ca. Ryanodine is thought to impair Ca-storing activity of caffeine-sensitive Ca stores by fixing Ca-induced Ca-release channels in the open state. We found that ryanodine increased sensitivity of taenia caecum against McN-A-343, AHR-602 or K, suggesting that the functional changes in intracellular Ca stores affect the contraction elicited by influx of extracellular Ca, and that desensitization is induced by increased Ca-storing activity of intracellular Ca stores. References Hishinuma, S. and Uchida, M.K., 1988, Br. J. Pharmacol., 94, 882. Hishinuma, S. and Uchida, M.K., 1989, Biochem. Biophys. Res. Commun., 162, 733. Shimada, S. and Uchida, M.K., 1988, Jap. J. Pharmacol., 46, 157P.
I P.fr.357 I
FMRF-NI-Iz-induced contraction might be resistant to organic Caz+-antagonists in molluscan smooth muscle Kizawa, Y., Inudoh, S., Sano *, M., Y a m a z a k i *, A., T s u k i m u r a *, T. and Murakami, H. Department of Physiology and Anatomy, Nihon University College of Pharmacy, Funabashi, Chiba 274 and * Department of Oral Physiology, School of Dentistry, Showa University, Shinagawa-ku, Tokyo 142, Japan
FMRF-amida (Phe-Met-Arg-Phe-NH2) had been initially found in ganglionic cells of Macrocallista nimbosa (Price , 1• .o'~7~ e t aJ., , , . r , .and the finding was then extended to other species (e.g., molluscan muscles and mammalian nervous systems). The amino acid sequence is same to the C-terminal region of enkephalin-related peptide (Tyr-Gly-Gly-PheMet-Arg-Phe-OH). Its physiological functions is not yet clear, but recently, some investigators suggested its function as neurotransmitter or hormone. FMRF-amide also contracts the anterior byssus retractor muscle (ABRM) of Mytilus edulis. However, few precise investigations for the phaxmacological and physiological responses to FMRF-amide has been reported. In the present study, a mechanism of action of FMRF-amide and immunohistochemical distribution of FMRF-amide-immunoreactivity have been investigated in the ABRM. Methods: Sea mussels, Mytilus edulis L., collected from Ise Bay (Japan) were used. The animals were stored in circulating artificial seawater at a temperature of about 10 ° C and used within a week after collection. Immunohistochemical distribution of FMRF-amide was determined using the methods of Williams and Dockray (1983). In mechanical response, muscle bundle (about 1 mm in diameter) dissected from ABRM was suspended in a 5 ml organ bath filled with artificial seawater, which was contained various concentrations of CaCI 2- The response was recorded isometrically under resting tension of 0.2 g. Results and discussion: FMRF-amide-immunoreactivity was determined on nerve fibers in the ABRM. On the other hand, FMRF-amide (10-7-10 -5 M) contracted the ABRM concentration-dependently. The concentration-response curve of FMRF-amide was rightward shifted by an analogue of FMRF-amide, FMRf-amide ([D-Phe4]FMRF amide, putative FMRF-amide receptor antagonist) in a parallel manner (pA e = 4.62 + 0.03). However, we failed to block the contractile response to FMRF-amide with organic Ca2+-antagonists ( > 10 -4 M, diltiazem; > 10 -4 M, verapamil; > 10 -5 M, nifedipine; > 10 -5 M, nicardipine; > 10 -6 M, o~-conotoxin GVIA and > 10 -4 M, TMB-8). On the contrary, inorganic Ca2+-antagonist, MnCI 2 reduced the contraction induced by FMRF-amide and 100 mM K+-solution. In the Ca2+-omitted solution (containing 2 mM EGTA), indeed, the contractile response to FMRF-amide was almost abolished. Therefore, FMRF-amide-induced contraction might be dependent on extracellular Ca-ions. On the other hand, 100 mM K+-solution also contracted the ABRM, and this response was
241S reduced/abolished by the above organic Ca2+-antagonists, except ~o-conotoxin GVIA. Results suggest that FMRFamide might be endogenous neuronal peptide, and that this peptide-induced contraction might be mediated through an action on FMRF-amide receptors and not be mediated through organic Ca ~+-antagonists-sensitive Ca2+-channels.
References D.A. Price and MJ. Greenberg, 1977, Science, 197, 670. R.G. Williams and GJ. Dockray, 1983, Brain Res., 276, 213.
Effects of PAF on smooth muscle in vitro Brandt, H.D. a n d Prinsloo, J.J. Department of Pharmacology and Therapeutics, Medunsa, P 0 Medunsa, 0204 South Africa
Platelet-activating-factor has been implicated in the pathogenesis of asthma. In the presence of blood platelets, it is involved in an as yet unknown way in the induction of bronchospasm, it is able to induce an inflammatory reaction of the late phase of asthma and it car, lead to a hyperreactivity of airway smooth muscle in vivo. If hyperreactivity could be shown in vitro, then it would be easier to investigate aspects of this phenomenon especially if mechanisms associated with hyperreactivity could be investigated in the same model. For this purpose we chose to use the isolated guinea-pig tracheal chain preparation suspended in a 10 ml organ bath in Krebs-Henseleit solution (KHS) (Van Rossum, 1963). The muscle was attached to a potentiometric strain gauge transducer registering impulses on a chart recorder. After suspension the muscle was allowed to equilibrate and stabiliTe for + 30 minutes and when a stable baseline was reached, it was recorded. A concentration of methacholine (MCh) which was determined in separate experiments to induce a maximal contraction (1 × 10-4M MCh) was then used to contract the muscle. The maximal recorded contraction was then taken as a 100~; contraction. The MCh was rinsed out with KHS and the muscle allowed to relax back to the previous baseline (+5~;). Viable human blood platelets were then added (1 × l0 s per ml of organ bath fluid). The added platelets had no significant effect on the baseline position. Immediately following this, a complete concentration-contraction response curve was done with PAF using half-log concentration increments from 1 × 10 -9 to 3 × 1 0 - t M . We showed that the smooth muscle contraction due to PAF is concentration dependant. The maximal contraction recorded v,as expressed as a percentage of the MCh contraction. The PAF and platelets were washed out with KHS and the muscle allowed to relax back to baseline ( + 5~;). After 45 minutes rest the muscle was contracted a second time with 1 × 10-4M MCh. This second MCh induced contraction was used to measure changes in sensitivity to MCh. A significant 234~ increase in height of contraction compared to the first MCh challenge was found. (In separate experiments the preparation was contracted using MCh alone in three consecutive contractions, but where no PAF exposure occurred. No significant difference in the contraction response was found). Thus the increased sensitivity to MCh was induced by the PAF exposure. In separate experiments it was shown that the smooth muscle contraction induced by PAF (in the presence of platelets) can be suppressed in a concentration dependant way by flurbiprofen, but the response cannot be suppressed totally. Using methysergide or mepyramine in separate experiments, both drugs indi~duaiiy can reduce the contraction response, but not totally. However, flurbiprofen, mepyramine and methysergide in combination can totally suppress the muscle contraction response. In contrast, these three drugs in combination cannot (at maximally effective concentrations), totally suppress the increase in reactivity towards MCh as measured by the second MCh exposure. A small but significant enhanced response to MCh remains evident. We conclude that PAF in the presence of blood platelets induces a concentration dependant airway smooth muscle contraction in the isolated guinea-pig trachea. This contraction can be totally prevented using a combination of methysergide, mepyramine and flurbiprofen. At the same time PAF induces an increased reactivity to MCh resulting in a 243~ increase in smooth muscle contraction. This increased reactivity cannot be totally prevented by the same three antagonists that completely blocked smooth muscle contraction induced by PAF. Thus at least one mechanism is involved in the hyperreactivity response which differs from those involved in PAF induced smooth muscle contraction.
agonists induced contractions only in the presence of extracellular Ca, and carbachol desensitized McN-A-343- or AHR-602-induced contraction. Desensitization by carbachol was also observed in K-induced contraction, believed to occur by Ca influx through the voltage-dependent Ca channels activated by X. It is possible that ca~bachol desensitizes the McN-A-343-, AHR-602- or K-induced contraction by suppressing the function of Ca channels in the plasma membrane. However, it is also possible that contraction due to McN-A-343, AHR-602 or K is influenceP by changes in intracellular Ca stores. Involvement of intracellular Ca stores in the contraction elicited influx of extracellular Ca. Ryanodine is thought to impair Ca-storing activity of caffeine-sensitive Ca stores by fixing Ca-induced Ca-release channels in the open state. We found that ryanodine increased sensitivity of taenia caecum against McN-A-343, AHR-602 or K, suggesting that the functional changes in intracellular Ca stores affect the contraction elicited by influx of extracellular Ca, and that desensitization is induced by increased Ca-storing activity of intracellular Ca stores. References Hishinuma, S. and Uchida, M.K., 1988, Br. J. Pharmacol., 94, 882. Hishinuma, S. and Uchida, M.K., 1989, Biochem. Biophys. Res. Commun., 162, 733. Shimada, S. and Uchida, M.K., 1988, Jap. J. Pharmacol., 46, 157P.
I P.fr.357 I
FMRF-NI-Iz-induced contraction might be resistant to organic Caz+-antagonists in molluscan smooth muscle Kizawa, Y., Inudoh, S., Sano *, M., Y a m a z a k i *, A., T s u k i m u r a *, T. and Murakami, H. Department of Physiology and Anatomy, Nihon University College of Pharmacy, Funabashi, Chiba 274 and * Department of Oral Physiology, School of Dentistry, Showa University, Shinagawa-ku, Tokyo 142, Japan
FMRF-amida (Phe-Met-Arg-Phe-NH2) had been initially found in ganglionic cells of Macrocallista nimbosa (Price , 1• .o'~7~ e t aJ., , , . r , .and the finding was then extended to other species (e.g., molluscan muscles and mammalian nervous systems). The amino acid sequence is same to the C-terminal region of enkephalin-related peptide (Tyr-Gly-Gly-PheMet-Arg-Phe-OH). Its physiological functions is not yet clear, but recently, some investigators suggested its function as neurotransmitter or hormone. FMRF-amide also contracts the anterior byssus retractor muscle (ABRM) of Mytilus edulis. However, few precise investigations for the phaxmacological and physiological responses to FMRF-amide has been reported. In the present study, a mechanism of action of FMRF-amide and immunohistochemical distribution of FMRF-amide-immunoreactivity have been investigated in the ABRM. Methods: Sea mussels, Mytilus edulis L., collected from Ise Bay (Japan) were used. The animals were stored in circulating artificial seawater at a temperature of about 10 ° C and used within a week after collection. Immunohistochemical distribution of FMRF-amide was determined using the methods of Williams and Dockray (1983). In mechanical response, muscle bundle (about 1 mm in diameter) dissected from ABRM was suspended in a 5 ml organ bath filled with artificial seawater, which was contained various concentrations of CaCI 2- The response was recorded isometrically under resting tension of 0.2 g. Results and discussion: FMRF-amide-immunoreactivity was determined on nerve fibers in the ABRM. On the other hand, FMRF-amide (10-7-10 -5 M) contracted the ABRM concentration-dependently. The concentration-response curve of FMRF-amide was rightward shifted by an analogue of FMRF-amide, FMRf-amide ([D-Phe4]FMRF amide, putative FMRF-amide receptor antagonist) in a parallel manner (pA e = 4.62 + 0.03). However, we failed to block the contractile response to FMRF-amide with organic Ca2+-antagonists ( > 10 -4 M, diltiazem; > 10 -4 M, verapamil; > 10 -5 M, nifedipine; > 10 -5 M, nicardipine; > 10 -6 M, o~-conotoxin GVIA and > 10 -4 M, TMB-8). On the contrary, inorganic Ca2+-antagonist, MnCI 2 reduced the contraction induced by FMRF-amide and 100 mM K+-solution. In the Ca2+-omitted solution (containing 2 mM EGTA), indeed, the contractile response to FMRF-amide was almost abolished. Therefore, FMRF-amide-induced contraction might be dependent on extracellular Ca-ions. On the other hand, 100 mM K+-solution also contracted the ABRM, and this response was
241S reduced/abolished by the above organic Ca2+-antagonists, except ~o-conotoxin GVIA. Results suggest that FMRFamide might be endogenous neuronal peptide, and that this peptide-induced contraction might be mediated through an action on FMRF-amide receptors and not be mediated through organic Ca ~+-antagonists-sensitive Ca2+-channels.
References D.A. Price and MJ. Greenberg, 1977, Science, 197, 670. R.G. Williams and GJ. Dockray, 1983, Brain Res., 276, 213.
Effects of PAF on smooth muscle in vitro Brandt, H.D. a n d Prinsloo, J.J. Department of Pharmacology and Therapeutics, Medunsa, P 0 Medunsa, 0204 South Africa
Platelet-activating-factor has been implicated in the pathogenesis of asthma. In the presence of blood platelets, it is involved in an as yet unknown way in the induction of bronchospasm, it is able to induce an inflammatory reaction of the late phase of asthma and it car, lead to a hyperreactivity of airway smooth muscle in vivo. If hyperreactivity could be shown in vitro, then it would be easier to investigate aspects of this phenomenon especially if mechanisms associated with hyperreactivity could be investigated in the same model. For this purpose we chose to use the isolated guinea-pig tracheal chain preparation suspended in a 10 ml organ bath in Krebs-Henseleit solution (KHS) (Van Rossum, 1963). The muscle was attached to a potentiometric strain gauge transducer registering impulses on a chart recorder. After suspension the muscle was allowed to equilibrate and stabiliTe for + 30 minutes and when a stable baseline was reached, it was recorded. A concentration of methacholine (MCh) which was determined in separate experiments to induce a maximal contraction (1 × 10-4M MCh) was then used to contract the muscle. The maximal recorded contraction was then taken as a 100~; contraction. The MCh was rinsed out with KHS and the muscle allowed to relax back to the previous baseline (+5~;). Viable human blood platelets were then added (1 × l0 s per ml of organ bath fluid). The added platelets had no significant effect on the baseline position. Immediately following this, a complete concentration-contraction response curve was done with PAF using half-log concentration increments from 1 × 10 -9 to 3 × 1 0 - t M . We showed that the smooth muscle contraction due to PAF is concentration dependant. The maximal contraction recorded v,as expressed as a percentage of the MCh contraction. The PAF and platelets were washed out with KHS and the muscle allowed to relax back to baseline ( + 5~;). After 45 minutes rest the muscle was contracted a second time with 1 × 10-4M MCh. This second MCh induced contraction was used to measure changes in sensitivity to MCh. A significant 234~ increase in height of contraction compared to the first MCh challenge was found. (In separate experiments the preparation was contracted using MCh alone in three consecutive contractions, but where no PAF exposure occurred. No significant difference in the contraction response was found). Thus the increased sensitivity to MCh was induced by the PAF exposure. In separate experiments it was shown that the smooth muscle contraction induced by PAF (in the presence of platelets) can be suppressed in a concentration dependant way by flurbiprofen, but the response cannot be suppressed totally. Using methysergide or mepyramine in separate experiments, both drugs indi~duaiiy can reduce the contraction response, but not totally. However, flurbiprofen, mepyramine and methysergide in combination can totally suppress the muscle contraction response. In contrast, these three drugs in combination cannot (at maximally effective concentrations), totally suppress the increase in reactivity towards MCh as measured by the second MCh exposure. A small but significant enhanced response to MCh remains evident. We conclude that PAF in the presence of blood platelets induces a concentration dependant airway smooth muscle contraction in the isolated guinea-pig trachea. This contraction can be totally prevented using a combination of methysergide, mepyramine and flurbiprofen. At the same time PAF induces an increased reactivity to MCh resulting in a 243~ increase in smooth muscle contraction. This increased reactivity cannot be totally prevented by the same three antagonists that completely blocked smooth muscle contraction induced by PAF. Thus at least one mechanism is involved in the hyperreactivity response which differs from those involved in PAF induced smooth muscle contraction.