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Tris buffer effects on melanophore aggregating responses
Camp.
Biochem.
Physiol.
Vol. 82C. No. 2. pp. 501-503,
1985
0306-44921135$3.00 + 0.00 ICY19x5 Pergamon Press Ltd
Printed in Great Britain
TRIS
BUFFER EFFECTS AGGREGATING
ON MELANOPHORE RESPONSES
MARIA A. VISCONTI and ANA M. L. CASTRUCCI Departamento
de Fisiologia,
Instituto
de Biocikncias, Universidade Brasil
de S%o Paula,
CP
11176.Sib Paula
(Received 21 March 1985)
Abstract-l. The adverse effects of tris (hydroxymethyl)-aminomethane (Tris) are for the first time reported on melanophore responses to agonists and potassium chloride. 2. Melanophore responses in Tris- or bicarbonate-buffered solutions were compared. 3. In the presence of Tris, the cumulative dose-response curve to norepinephrine was significantly shifted to the left. whereas methoxamine dose-response curves were similar in both buffers. 4. The percentage aggregation in response to synthetic MCH (melanin concentrating hormone) was not affected by Tris in the bathing medium. 5. The cumulative dose-response curve to potassium chloride was leftward shifted (one log case) in Tris-buffered solution. 6. These results suggest that in fish melanophore preparations Tris might exert its actions on the presynaptic membrane and/or on the synaptic cleft enzyme COMT, drawing on a greater availability of neurotransmitters at the melanophore membrane receptors.
INTRODUCTION
MATERIALS AND METHODS
Tris (hydroxymethyl)-aminomethane (Tris) has been widely employed as a buffering agent for physiological solutions in various muscle preparations. It is also the common buffer used for pharmacological studies on fish chromatophores. However, Tris has been reported to adversely affect contractile responses to agonists. Gillespie and McKnight (1976) observed an inhibitory effect of Tris on motor responses to adrenergic nerve stimulation in rat anococcygeus and vas deferens, and in rabbit ear artery, though the sensitivity of the latter preparation to norepinephrine was enhanced. Furthermore, an increase or reduction in tonus, depending on the type of muscle, was also observed in Tris solutions. Turlapaty et al. (1978) demonstrated that Tris attenuated epinephrine, angiotensin and potassium induced contractile responses in rat portal vein and aorta, as well as abolishing aorta spontaneous activity. Johansson et al. (1979) and Karaki et al. (1981) did not observe such effects of Tris on vascular and intestinal smooth muscle preparations. These authors proposed that the reported altered results would probably be due to different experimental conditions and/or artifacts. Nevertheless, recent studies have confirmed the adverse effects of Tris on smooth muscle contractility (Kwan and Daniel, 1981; Altura et al., 1982), and Tris was suggested to interfere with the handling of Ca2 + by the muscle. Conclusive results were reported by Kwan et al. (1984), demonstrating that Tris inhibits Ca*+ binding and active transport in microsomal membrane fractions from rat vas deferens. In an attempt to delineate appropriate experimental procedures to study ionic requirements for agonist action on fish melanophores, the present results with Tris and bicarbonate buffered solutions were obtained.
All experiments were performed with dermal melanophores from adult Poecilia reticulata (a freshwater teleostean), raised in our laboratory. Dorsolateral scales were excised and immediately immersed in physiological salt solution (PSS). The PSS was in mM as follows: NaCl, 128; KCl, 2.7; CaCl,. 1.8; glucose 5.6. The bicarbonate-buffered PSS contained 2.5 mM NaHCO,, and the pH (7.2-7.4) was adjusted with 954: 02-5y, CO,. In the Tris-buffered PSS, bicarbonate was replaced with 5 mM Tris, and the pH (7.2-7.4) adjusted with 1.O N HCl. All the experiments were carried out at 22-25 ‘C. One scale was then fixed, epidermis side up, to a cover slip, which was turned down and mounted in a perfusion chamber. The chamber was filled up with PSS or agonist solutions. which were diluted in PSS immediately prior to use. The melanophore responses were recorded as the percentage change in apparent length of a given cellular process, measured with the aid of an ocular micrometer. Full melanophore dispersion was considered to be settled after a 30 min equilibration in PSS and maximal aggregation after either a high potassium solution (Nagahama. 1953) or lO_‘M norepinephrine treatment. Cumulative dose-response curves (DRC) were determined for each agonist by increasing the bath concentration stepwise in a log scale (van Rossum and van den Brink, 1963). Since the chamber was kept sealed during the experiment, the bathing solution was switched by the next one, after equilibration was attained for each drug concentration. This procedure was repeated until the maximal response of each preparation to a given agonist was achieved. The ED,, value (agonist dose eliciting 500/” of maximal response) was calculated as the geometric mean (with the respective 95”” confidence interval) of equieffective doses from single DRC (Fleming ef ol., 1972). The results were compared using Student’s f test and analysis of variance. The following chemicals were used: I-norepinephrine (NE), dl-methoxamine hydrochloride and tris (hydroxymethyl)-aminomethnane (Tris) from Sigma Co. and melanin concentrating hormone (MCH), synthesized and purified by B. C. Wilkes in the V. J. Hruby laboratory at the University of Arizona. 501
502 Table 1. Effects of Tris buffer on sympathomimetic amine and potassium induced aggregating responses in fish melanophores, as assessed by ED,, values and their 95% confidence intervals Agonist
Tris buffer
Bicarbonate buffer
Norepinephrine
3.44 x lo-‘M (2.23%5.31)* (iv = 8) 1.67 x IO-‘M (1.05-2.63) [N =?) 1.58 x IO-’ M (t&--1.67)* fN=5)
12.34 x IO-‘M (10.56-14.1 I) (N = 10) 3.23 x IO-‘M (1,49-6.971 (W = 7) 13.39 x 10-r M (I 1,98-14.80) fN=5)
Methoxamine Potassium
*Signilicantiy different from those obtained in bicarbonate-buffered
Table 2. Meianophore aggregating responses to melanin-concentrating hormone (MCH), in Tris and bicarbonate buffers, recorded at different time oeriods Time (min) 5
IQ 15
Tris buffer
Bicarbonate butfer (N = 1t)f
54.4 * 41.8 93.5 & 14.4 98.8 + 2.4
55.2 f 40.3 92.5 + 14.5 99.8 & 0.7
*Values represent the mean (&SE) percentage of aggregation
RESULTS
Table 1 shows the potentiation (as assessed by EL&) of nore~~ne~hrine (NE) and potassium chloride (&Xl) aggregating effects on melanophores in T&buffered solution as compared to bicarbonate solution. In contrast, the cumulative doseresponse curve to methoxamine was not altered by Tris. Figure 1 demonstrates that Tris significantly lowered the ED,, values for both NE and KC& though the leftward shift of the potassium dose-response curve was much more pronounced. The threshold concentrations of both NE and KCI were not affected in the presence of Tris {Fig. 1). The magnitude of maximal aggregating response to any of the agonists did not change in Tris-buffered solution. Experiments with the putative fish hormone MCI-I revealed that the melanophore responses to this agonist (recorded as percentage aggregation over
different incubation (Table 2).
solutions (P c 0.05).
periods) were not altered by Tris
The impaired agonist-induced contractile reSIX.XW~~ of smooth and cardiac muscles in the presence of Tris have been attributed to various possible actions of this buffer. Despite some controversial results obtained in different laboratories, there seems to be a common agreement that Tris (1) increases the intracellular pII, which might explain the potentiation abserved in some agonist-induced responses {reduction of ED,) (Gillespie and McKnight, 1976) and (2) inhibits binding and active transport of Ca’ + , probably resufting in the reduced responses in smooth muscles reported by most authors (Gillespie and McKnight, 1976; Turlapaty et al., 1978; Kwan and Daniel, 1981; Altura et ul., 1982; Kwan et al., 1984)” In melanophore preparations, the presence of Tris as the buffering agent potentiated the aggregating responses to NE and KC1 but did not affect those to methoxamine and MCH. Therefore, the effects of Tris are specific, depending upon the agonist employed. In this way, the possibility that Tris directly affects the effector responses through an increase in intracellular alkalinity may be discarded. An influx of Ca2+ was reported to take place during melanosome aggregation (Negishi and Obika. 1983). If Tris exerted its action on melanophore Ca” +
Fig. 1. Effects of Tris buffer on norepinephrine (XE) curves in fish melanophores. Each value represents
and potassium (KG) cumniative dose-response the mean (*SE)
percentage
of aggregation.
Tris affects melanophore responses
transport, an inhibition of the aggregating response rather than a potentiation would be expected. In addition, Tris would affect the aggregating responses to all the adrenergic agonists and that was not the case. Although both NE and methoxamine are adrenomimetic agents acting through a-receptors, only the former had its activity enhanced by Tris. Considering that NE is taken up by the nerve endings, whereas methoxamine, as a nonphysiological agonist. is not (Trendenlenburg et al., 1970), Tris might be acting on the pre-synaptic membrane, thus affecting only the amount of available NE. On the basis of the above consideration, one should also expect a leftward shift of the KC1 dose-response curve, as we actually obtained, since it induces the release of the aggregating neurotransmitter from the nerve endings (for references see Kumazawa and Fujii, 1984). As is already known, cocaine has been widely used as a neuronal uptake blocker. Therefore it would be an useful pharmacological tool to confirm the hypothesis above. One might expect that, in the presence of cocaine, either the NE or the KCI dose-response curve would be shifted to the left in both Tris- and bicarbonate-buffered solutions. But, since Tris would be acting SynergisticalIy with cocaine in inhibiting NE uptake, the displacement of the curves should be much more pronounced in Tris solutions. However, in fish melanophore preparations, this approach proved to be difficult since cocaine has an intrinsic aggregating activity (Visconti and Castrucci, unpublished data). Tris might also be inhibiting the synaptic enzyme COMT (catechoi-0-methyltransferase) but the degradation rate of NE promoted by this enzyme has been reported to be so low in melanophores (Katayama and Yamada, 1983) that its inhibition would hardly be the key event to allow for the marked increase in NE and KC1 potencies. Based on these considerations it is suggested that Tris probably alters the membrane properties of the nerve endings present in the fish melanophore preparations. As a result, a decrease in neuronal uptake seems to take place, thus enhancing the availability of NE. In the case of KCl, an increase in neurotransmitter retease might still be occurring, causing the more pronounced leftward shift of its dose-response curve as compared to NE. Acknowfledgemenfs-This
work was supported by FAPESP (grants 84/1967-X and 84/1263-O. Zoologia). We are thank-
503
ful to Drs V. J. Hruby, M. E. Hadiey and B. C. Wilkes for the generous gift of MCH and to Dr R. Saban for methoxamine. REFERENCES Altura B. M., AItura B. T., Carella A. and Turlapaty P. D. M. V. (1982) Cal- coupling in vascular smooth muscle: MgL’ and buffer effects on contractility and membrane Ca’+ movements, Can. .I. Physial. Pharmac. 60,459-482. Fleming W. W., Westfall D. P., de la Lande I. S. and Jellet L. B. (1972) Log-normal distribution of eauieffective doses of norepiiephrine and acetylcholine &in several tissues. J. Pharmac. exp. Ther. 181, 339-345. Gillespie J. S. and McKnight A. T. (1976) Adverse effects of Tris hydrochloride, a commonly used buffer in physiofogicai media. J. Physiof. 259, 581-573. Johansson B,. Liung B.. Hellstrand P., Uvehus B. and Sigurdsson S. B. (1579) Effects of Tris on vascular smooth muscle (letter to the editor). Am. .I. Ph.vsioI. 237, W410H411.
Karaki H., Suzuki T. and Urakawa N. (1981) Tris does not inhibit isolated vascular or intestinal smooth muscle contraction. Am. J. Phvsiol. 241. H337-H341. Katayama H. and Yamada K. (1983) Effect of pyrogallol on responses of melanophores to norepinephrine and KC1 on isolated caudal tins of goby, Tridentiger trigonocephalus. J. Sri. ~~Fas~~~~aUnio. Ser. B, Dir?. 1 31, 127-136. Kumazawa T. and Fujii R. (1984) Concurrent releases of norepincph~ne and purines by potassium from adrenergic melanosome aggregating nerve in tilapia. Camp. B&hem.
Physiol. 78?!, 263..-266.
Kwan C. Y. and Daniel E. E. (1981) Tris inhibits calcium accumulation by plasma membrane fractions isolated from vascular smooth muscles. Biochem. int. 2, 429-436. Kwan C. Y., Sakai Y. and Daniel E. E. (1984) Tris inhibits binding and transport of calcium in microsomal fraction isolated from rat vas deferens. Arch. ht. Pha~rnac~~d~J~, 269, 252-262.
Nagahama H. (1953) Action of potassium ions on the melanophores in an isolated fish scale. fap. f. 2002. 11, 75-85. Negishi S. and Obika M. (1983) The role of calcium and magnesium on pigment translocation in melanophores of Oryzias latipes. Abstracts from the XIIth International Pigment Cell Conference, p. 163. Trendenlenburg U., Maxwell R. A. and Pluchino S. (1970) Methoxamine as a tool to assess the importance of intraneuronal uptake of ~-norepineph~ne in the cat’s nictating membrane. J. Phurmac. exp. Ther. 172, 91-99. Turlapaty P. D. M. V.. Altura 8. T. and Altura B. M. (I 978) Influence of Tris on contractile responses of isolated rat aorta and portal vein. Am. J. Physiol. 235, H208-H213. van Rossum J. M. and van den Brink F. G. (1963) Cumulative dose-response curves. I. Introduction to the technique. Arch. int. Pharmaco&n. 143, 240-246.
Biochem.
Physiol.
Vol. 82C. No. 2. pp. 501-503,
1985
0306-44921135$3.00 + 0.00 ICY19x5 Pergamon Press Ltd
Printed in Great Britain
TRIS
BUFFER EFFECTS AGGREGATING
ON MELANOPHORE RESPONSES
MARIA A. VISCONTI and ANA M. L. CASTRUCCI Departamento
de Fisiologia,
Instituto
de Biocikncias, Universidade Brasil
de S%o Paula,
CP
11176.Sib Paula
(Received 21 March 1985)
Abstract-l. The adverse effects of tris (hydroxymethyl)-aminomethane (Tris) are for the first time reported on melanophore responses to agonists and potassium chloride. 2. Melanophore responses in Tris- or bicarbonate-buffered solutions were compared. 3. In the presence of Tris, the cumulative dose-response curve to norepinephrine was significantly shifted to the left. whereas methoxamine dose-response curves were similar in both buffers. 4. The percentage aggregation in response to synthetic MCH (melanin concentrating hormone) was not affected by Tris in the bathing medium. 5. The cumulative dose-response curve to potassium chloride was leftward shifted (one log case) in Tris-buffered solution. 6. These results suggest that in fish melanophore preparations Tris might exert its actions on the presynaptic membrane and/or on the synaptic cleft enzyme COMT, drawing on a greater availability of neurotransmitters at the melanophore membrane receptors.
INTRODUCTION
MATERIALS AND METHODS
Tris (hydroxymethyl)-aminomethane (Tris) has been widely employed as a buffering agent for physiological solutions in various muscle preparations. It is also the common buffer used for pharmacological studies on fish chromatophores. However, Tris has been reported to adversely affect contractile responses to agonists. Gillespie and McKnight (1976) observed an inhibitory effect of Tris on motor responses to adrenergic nerve stimulation in rat anococcygeus and vas deferens, and in rabbit ear artery, though the sensitivity of the latter preparation to norepinephrine was enhanced. Furthermore, an increase or reduction in tonus, depending on the type of muscle, was also observed in Tris solutions. Turlapaty et al. (1978) demonstrated that Tris attenuated epinephrine, angiotensin and potassium induced contractile responses in rat portal vein and aorta, as well as abolishing aorta spontaneous activity. Johansson et al. (1979) and Karaki et al. (1981) did not observe such effects of Tris on vascular and intestinal smooth muscle preparations. These authors proposed that the reported altered results would probably be due to different experimental conditions and/or artifacts. Nevertheless, recent studies have confirmed the adverse effects of Tris on smooth muscle contractility (Kwan and Daniel, 1981; Altura et al., 1982), and Tris was suggested to interfere with the handling of Ca2 + by the muscle. Conclusive results were reported by Kwan et al. (1984), demonstrating that Tris inhibits Ca*+ binding and active transport in microsomal membrane fractions from rat vas deferens. In an attempt to delineate appropriate experimental procedures to study ionic requirements for agonist action on fish melanophores, the present results with Tris and bicarbonate buffered solutions were obtained.
All experiments were performed with dermal melanophores from adult Poecilia reticulata (a freshwater teleostean), raised in our laboratory. Dorsolateral scales were excised and immediately immersed in physiological salt solution (PSS). The PSS was in mM as follows: NaCl, 128; KCl, 2.7; CaCl,. 1.8; glucose 5.6. The bicarbonate-buffered PSS contained 2.5 mM NaHCO,, and the pH (7.2-7.4) was adjusted with 954: 02-5y, CO,. In the Tris-buffered PSS, bicarbonate was replaced with 5 mM Tris, and the pH (7.2-7.4) adjusted with 1.O N HCl. All the experiments were carried out at 22-25 ‘C. One scale was then fixed, epidermis side up, to a cover slip, which was turned down and mounted in a perfusion chamber. The chamber was filled up with PSS or agonist solutions. which were diluted in PSS immediately prior to use. The melanophore responses were recorded as the percentage change in apparent length of a given cellular process, measured with the aid of an ocular micrometer. Full melanophore dispersion was considered to be settled after a 30 min equilibration in PSS and maximal aggregation after either a high potassium solution (Nagahama. 1953) or lO_‘M norepinephrine treatment. Cumulative dose-response curves (DRC) were determined for each agonist by increasing the bath concentration stepwise in a log scale (van Rossum and van den Brink, 1963). Since the chamber was kept sealed during the experiment, the bathing solution was switched by the next one, after equilibration was attained for each drug concentration. This procedure was repeated until the maximal response of each preparation to a given agonist was achieved. The ED,, value (agonist dose eliciting 500/” of maximal response) was calculated as the geometric mean (with the respective 95”” confidence interval) of equieffective doses from single DRC (Fleming ef ol., 1972). The results were compared using Student’s f test and analysis of variance. The following chemicals were used: I-norepinephrine (NE), dl-methoxamine hydrochloride and tris (hydroxymethyl)-aminomethnane (Tris) from Sigma Co. and melanin concentrating hormone (MCH), synthesized and purified by B. C. Wilkes in the V. J. Hruby laboratory at the University of Arizona. 501
502 Table 1. Effects of Tris buffer on sympathomimetic amine and potassium induced aggregating responses in fish melanophores, as assessed by ED,, values and their 95% confidence intervals Agonist
Tris buffer
Bicarbonate buffer
Norepinephrine
3.44 x lo-‘M (2.23%5.31)* (iv = 8) 1.67 x IO-‘M (1.05-2.63) [N =?) 1.58 x IO-’ M (t&--1.67)* fN=5)
12.34 x IO-‘M (10.56-14.1 I) (N = 10) 3.23 x IO-‘M (1,49-6.971 (W = 7) 13.39 x 10-r M (I 1,98-14.80) fN=5)
Methoxamine Potassium
*Signilicantiy different from those obtained in bicarbonate-buffered
Table 2. Meianophore aggregating responses to melanin-concentrating hormone (MCH), in Tris and bicarbonate buffers, recorded at different time oeriods Time (min) 5
IQ 15
Tris buffer
Bicarbonate butfer (N = 1t)f
54.4 * 41.8 93.5 & 14.4 98.8 + 2.4
55.2 f 40.3 92.5 + 14.5 99.8 & 0.7
*Values represent the mean (&SE) percentage of aggregation
RESULTS
Table 1 shows the potentiation (as assessed by EL&) of nore~~ne~hrine (NE) and potassium chloride (&Xl) aggregating effects on melanophores in T&buffered solution as compared to bicarbonate solution. In contrast, the cumulative doseresponse curve to methoxamine was not altered by Tris. Figure 1 demonstrates that Tris significantly lowered the ED,, values for both NE and KC& though the leftward shift of the potassium dose-response curve was much more pronounced. The threshold concentrations of both NE and KCI were not affected in the presence of Tris {Fig. 1). The magnitude of maximal aggregating response to any of the agonists did not change in Tris-buffered solution. Experiments with the putative fish hormone MCI-I revealed that the melanophore responses to this agonist (recorded as percentage aggregation over
different incubation (Table 2).
solutions (P c 0.05).
periods) were not altered by Tris
The impaired agonist-induced contractile reSIX.XW~~ of smooth and cardiac muscles in the presence of Tris have been attributed to various possible actions of this buffer. Despite some controversial results obtained in different laboratories, there seems to be a common agreement that Tris (1) increases the intracellular pII, which might explain the potentiation abserved in some agonist-induced responses {reduction of ED,) (Gillespie and McKnight, 1976) and (2) inhibits binding and active transport of Ca’ + , probably resufting in the reduced responses in smooth muscles reported by most authors (Gillespie and McKnight, 1976; Turlapaty et al., 1978; Kwan and Daniel, 1981; Altura et ul., 1982; Kwan et al., 1984)” In melanophore preparations, the presence of Tris as the buffering agent potentiated the aggregating responses to NE and KC1 but did not affect those to methoxamine and MCH. Therefore, the effects of Tris are specific, depending upon the agonist employed. In this way, the possibility that Tris directly affects the effector responses through an increase in intracellular alkalinity may be discarded. An influx of Ca2+ was reported to take place during melanosome aggregation (Negishi and Obika. 1983). If Tris exerted its action on melanophore Ca” +
Fig. 1. Effects of Tris buffer on norepinephrine (XE) curves in fish melanophores. Each value represents
and potassium (KG) cumniative dose-response the mean (*SE)
percentage
of aggregation.
Tris affects melanophore responses
transport, an inhibition of the aggregating response rather than a potentiation would be expected. In addition, Tris would affect the aggregating responses to all the adrenergic agonists and that was not the case. Although both NE and methoxamine are adrenomimetic agents acting through a-receptors, only the former had its activity enhanced by Tris. Considering that NE is taken up by the nerve endings, whereas methoxamine, as a nonphysiological agonist. is not (Trendenlenburg et al., 1970), Tris might be acting on the pre-synaptic membrane, thus affecting only the amount of available NE. On the basis of the above consideration, one should also expect a leftward shift of the KC1 dose-response curve, as we actually obtained, since it induces the release of the aggregating neurotransmitter from the nerve endings (for references see Kumazawa and Fujii, 1984). As is already known, cocaine has been widely used as a neuronal uptake blocker. Therefore it would be an useful pharmacological tool to confirm the hypothesis above. One might expect that, in the presence of cocaine, either the NE or the KCI dose-response curve would be shifted to the left in both Tris- and bicarbonate-buffered solutions. But, since Tris would be acting SynergisticalIy with cocaine in inhibiting NE uptake, the displacement of the curves should be much more pronounced in Tris solutions. However, in fish melanophore preparations, this approach proved to be difficult since cocaine has an intrinsic aggregating activity (Visconti and Castrucci, unpublished data). Tris might also be inhibiting the synaptic enzyme COMT (catechoi-0-methyltransferase) but the degradation rate of NE promoted by this enzyme has been reported to be so low in melanophores (Katayama and Yamada, 1983) that its inhibition would hardly be the key event to allow for the marked increase in NE and KC1 potencies. Based on these considerations it is suggested that Tris probably alters the membrane properties of the nerve endings present in the fish melanophore preparations. As a result, a decrease in neuronal uptake seems to take place, thus enhancing the availability of NE. In the case of KCl, an increase in neurotransmitter retease might still be occurring, causing the more pronounced leftward shift of its dose-response curve as compared to NE. Acknowfledgemenfs-This
work was supported by FAPESP (grants 84/1967-X and 84/1263-O. Zoologia). We are thank-
503
ful to Drs V. J. Hruby, M. E. Hadiey and B. C. Wilkes for the generous gift of MCH and to Dr R. Saban for methoxamine. REFERENCES Altura B. M., AItura B. T., Carella A. and Turlapaty P. D. M. V. (1982) Cal- coupling in vascular smooth muscle: MgL’ and buffer effects on contractility and membrane Ca’+ movements, Can. .I. Physial. Pharmac. 60,459-482. Fleming W. W., Westfall D. P., de la Lande I. S. and Jellet L. B. (1972) Log-normal distribution of eauieffective doses of norepiiephrine and acetylcholine &in several tissues. J. Pharmac. exp. Ther. 181, 339-345. Gillespie J. S. and McKnight A. T. (1976) Adverse effects of Tris hydrochloride, a commonly used buffer in physiofogicai media. J. Physiof. 259, 581-573. Johansson B,. Liung B.. Hellstrand P., Uvehus B. and Sigurdsson S. B. (1579) Effects of Tris on vascular smooth muscle (letter to the editor). Am. .I. Ph.vsioI. 237, W410H411.
Karaki H., Suzuki T. and Urakawa N. (1981) Tris does not inhibit isolated vascular or intestinal smooth muscle contraction. Am. J. Phvsiol. 241. H337-H341. Katayama H. and Yamada K. (1983) Effect of pyrogallol on responses of melanophores to norepinephrine and KC1 on isolated caudal tins of goby, Tridentiger trigonocephalus. J. Sri. ~~Fas~~~~aUnio. Ser. B, Dir?. 1 31, 127-136. Kumazawa T. and Fujii R. (1984) Concurrent releases of norepincph~ne and purines by potassium from adrenergic melanosome aggregating nerve in tilapia. Camp. B&hem.
Physiol. 78?!, 263..-266.
Kwan C. Y. and Daniel E. E. (1981) Tris inhibits calcium accumulation by plasma membrane fractions isolated from vascular smooth muscles. Biochem. int. 2, 429-436. Kwan C. Y., Sakai Y. and Daniel E. E. (1984) Tris inhibits binding and transport of calcium in microsomal fraction isolated from rat vas deferens. Arch. ht. Pha~rnac~~d~J~, 269, 252-262.
Nagahama H. (1953) Action of potassium ions on the melanophores in an isolated fish scale. fap. f. 2002. 11, 75-85. Negishi S. and Obika M. (1983) The role of calcium and magnesium on pigment translocation in melanophores of Oryzias latipes. Abstracts from the XIIth International Pigment Cell Conference, p. 163. Trendenlenburg U., Maxwell R. A. and Pluchino S. (1970) Methoxamine as a tool to assess the importance of intraneuronal uptake of ~-norepineph~ne in the cat’s nictating membrane. J. Phurmac. exp. Ther. 172, 91-99. Turlapaty P. D. M. V.. Altura 8. T. and Altura B. M. (I 978) Influence of Tris on contractile responses of isolated rat aorta and portal vein. Am. J. Physiol. 235, H208-H213. van Rossum J. M. and van den Brink F. G. (1963) Cumulative dose-response curves. I. Introduction to the technique. Arch. int. Pharmaco&n. 143, 240-246.