Biochimica et Biophysica Acta. 1014 (1989) 207-209
`Elsevier
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`BBAMCR 10245
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`BBAReport
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`207
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`Nicardipine as a Ca2+ channel blocker in single barnacle
`muscle fibers
`
`Huiwen Xie and E. Edward Bittar
`Department of Physiology, University of Wisconsin, Madison, WI (U.S.A.)
`
`(Received 10 May 1989)
`
`Key words: Nicardipine; Verapamil; Calcium ion channel; (Barnacle muscle fiber)
`
`A study has been made of the efficacy of nicardipine as a Ca2+ channel blocker by determining the magnitude of its
`effect on the stimulatory response of the ouabain-insensitive Na + efflux in single barnacle muscle fibers to 100 mM
`external K +, The results show ~hat nicardlpine (at pH 6.5) is a potent inhibitor, the minimal effective concentration
`being approx. 10 _., M and the IC!IO about 5 •10 _, M. Nicardipine, however, is not as potent as verapamil (at pH 6.5)
`on an equimolar basis. This is explained by assuming that the number of dihydropyridine receptors in the t-tubule
`membnmes of barnacle fibers is not high or that verapamil is able to block the sarcoplasmic retiwlum Ca2+ release
`channel in addition to the voltage-dependent ea2+ channels.
`
`Calcium channel blockers fall into two main classes:
`the dihydropyridine (DHP) and the non-DHP class. An
`example of the former is nifedipine or nicardipine, and
`of the latter verapamil or diltiazem. These drugs are
`known to block L-type, and not the T-orN-type Ca2+
`channel by binding to the a 1-subunit of the channel
`protein, a rather unique polypeptide of Mr 170 000,
`which has e sequence and membrane topology similar
`to that of the voltage-dependent Na + channel [1-5).
`Since the t-tubule membrane of skeletal muscle is the
`richest source of DHP receptors [6,7], skeletal muscle
`has proven to be a remarkably useful experimental
`preparation for various studies of the Ca 2+ channel,
`including its identification and purification.
`The work of Hagiwara and coworkers, e.g., Hagiwara
`[8] has shown that the action potential in barnacle fibers
`is Ca2+. rather than Na+-dependent, and that the volt(cid:173)
`age-dependent Ca2 + channels can be readily activated
`by depolarizing the fiber membrane, e.g., with high
`external K + (K; ), and inactivated by various agents,
`including verapamil. Depolarization of the barnacle
`muscle fibers membrane with high K; is also known to
`stimulate the ouabain-insensitive Na +efflux temporari(cid:173)
`ly by a mechanism which is readily stopped by prior
`external application of verapamil, injection of EGTA or
`
`Abbreviations: DHP, dihydropyridine; ASW, artificial sea water.
`
`Correspondence: E.E. Biuar, Department of Physiology, University of
`Wisconsin, 1300 University Avenue, Madison, WI 53'106, U.S.A.
`
`removal of external calcium [9]. These observations, in
`combination with other lines of evidence, have led to
`the conclusion that the observed rise in Na + efflux is
`due to a fall in myoplasmic pCa, resulting from the
`activation of volt&ge-dependent Ca2 + channels [10].
`Since verapamil has thus far been used as the standard
`Ca2 + channel blocker in this laboratory, it seemed a
`matter of special interest to examine the DHP class of
`drugs, and the derivative chosen in this instance for
`such testing is nicardipine. This communication de(cid:173)
`scribes experiments carried out using nicardipine with
`the main aim of determining whether it acts as a blocker
`in barnacle muscle fibers and whether it is as effective
`as verapamil.
`Specimens of the barnacle, Balanus nubilus were the
`source of single muscle fibers. The methods and proce(cid:173)
`dures used were essentially the same as those described
`elsewhere [10]. The artificial sea water (ASW) used as
`bathing solution contained 10 mM K+. ASW containing
`100 mM K+ was prepared by lowering NaCl in osmoti(cid:173)
`cally equivalent amounts. All bathing solutions used
`contained 10 mM imidazole as buffer in lieu of HC03,
`with the pH adjusted to 6.5. This was necessary because
`nicardipine has the following solubility profile: in only
`water (at 25°C), 8 ·10- 6 M at pH 7.4 (using 0.05 M
`phosphate as buffer) and approx. 10- 2 M at pH 4.6.
`Nicardipine in solution is known to be degraded by
`light but only about 2% of it undergoes breakdown over
`a 24 h period at a temperature of 30 ° C. (Information
`from Syntex Research, Palo Alto, CA.) Hence the ex(cid:173)
`periments described here were not carried out in the
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`0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
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`208
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`Fig. 1. Upper panel: the response or the ouabain-insensitive Na +
`ernux following a sudden lO.fold increase inK; in the presence of
`10-4 M nicardipine (rate constant for 22 Na efflux plot). Lower panel:
`the response of the ouabain-insensitive Na + efRux to a sudden 10-fold
`increase in Kt. Note that the time-frame of such control experiments
`is short, since the main object is to determine the magnitude of the
`stimulatory response. The rule is that the ouabain-insensitive Na+
`efflux returns to its original baseline [9].
`
`dark. All experiments were performed at room tempera(cid:173)
`ture 23 ± 1 o C. Changes in the ouabain-insensitive Na +
`efflux were estimated on the basis of the rate constant
`for 22Na efflux plots, and expressed as a percentage
`change (mean ± S.E. of mean) of the efflux. Ouabain,
`imidazole and Hepes were purchased from Sigma
`Chemical Company, St. Louis, MO. Nicardipine HCl
`was a gift from Syntex Research, Palo Alto, CA. ( ± )(cid:173)
`Verapamil HCl was obtained from Knoll Pharmaceuti(cid:173)
`cal Company, Whippany, NJ.
`In the first group of experiments, fibers suspended in
`10 mM imidazole/ ASW (pH 6.5) were treated with
`to-• M ouabain and 10-4 M nicardipine before de(cid:173)
`polarization by suddenly raising K; 10-fold (i.e., from
`10 to 100 mM). Companion controls were not exposed
`to nicardipine. The results of these experiments show
`that nicardipine causes a marked reduction in the mag(cid:173)
`nitude of the stimulatory response of the ouabain-insen(cid:173)
`sitive Na + efflux to high K!. A representative experi(cid:173)
`ment is presented in Fig. 1, where it can be seen that
`whereas external application of 10-4 M nicardipine is
`without effect on the resting ouabain-insensitive N a+
`efflux, it drastically reduces the size of the response to
`100 mM K~ (upper panel). Notice too that the residual
`response fails to decay completely, a situation that
`differs from that in the companion control fiber (lower
`panel); that is, the response of fibers exposed to high
`K + is rapid both in its full development and decay, with
`a return of the ouabain-insensitive Na + efflux to its
`original baseline level (e.g., Ref. 9). The magnitude of
`
`O
`
`5
`6
`7
`CD
`I'
`-LOG MOLAR NICARDIPINE CONCENTRATION
`Fig. 2. Concentration-response curve for the inhibitory action of
`nicardipine on the response of the ouabain-insensitive Na + efflux to
`100 mM K;. Vertical bars indicate±S.E.M. Each plotted point is the
`mean value of three determinations. All fibers were isolated from the
`same barnacle specimen.
`
`4
`
`the response in the presence of 10- 4 M nicardipine
`averages ·162 ± 24$ ( n == 19), as compared with 550 ±
`54% (n = 10) in controls (P < 0.001). To investigate this
`effect more closely, a concentration-response curve was
`determined. Shown in Fig. 2 is that the minimal effec(cid:173)
`tive concentration of nicardipine is roughly 10-7 M and
`that the concentration required to reduce the size of the
`response to 100 mM K; by 50% (i.e., the IC50 ) is
`4.5 ·10- 6 M (log scale). A threshold concentration of
`10-7 M is an interesting finding, particularly since it
`approaches the plasma concentration of nicardipine
`measured in healthy human volunteers receiving ther(cid:173)
`apeutic doses of nicardipine orally [11 ].
`Since this IC50 is about 10-times that for verapamil
`[9], and since the pKa of nicardipine and verapamil is
`7.2 and 8.5, respectively, it seemed important to com(cid:173)
`pare their efficacy using ASW at a low pH, e.g., 6.5. As
`
`ri0"4 M·VERAPAMIL~
`
`. - - - - IOOmM-K+
`
`lr---- 10"4 M·OUABAIN - - - - - . . ,1
`1
`..... ~ !
`..... .
`
`,_.161+1~~··~·~-~IMMI~~~ . . . . ~ ... I-1
`
`- -
`10"4 M·OUABAIN - - - - - .
`r 10"4 M·NICARDIPINEJ.
`1.
`+
`~ !
`1 roomM·K ~
`
`1
`
`Fig. 3. Upper panel: almost complete abolition of the response of the
`
`ouabain-insensitive Na+ efflux to 100 mM K: by to- 4 M verapamil.
`Lower panel: the response of the ouabain-insensitive Na + efflux to
`100 mM K: in the presence of 10-4 M nicardipine. Note that
`in both instances, verapamil and nicardipine were omitted at
`t=140min.
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`illustrated in Fig. 3, 10- 4 M verapamil almost com(cid:173)
`pletely abolishes the response of the ouabain-insensitive
`Na+ efflux to 100 mM K; (a residual response which
`averages 64 ± 9%, n = 8), whereas 10-4 M nicardipine
`is less effective as a blocker (a residual response which
`averages 162 ± 24%, n = 19). The difference in the two
`sizes is significant, P being < 0.01.
`The message which emerges is that nicardipine is less
`effective than verapamil as a Ca2 + channel blocker.
`However, this conclusion is based only on experiments
`that involve the monitoring of the ouabain-insensitive
`Na + efflux before and after suddenly raising the exter(cid:173)
`nal K. + concentration. The reason why nicardipine is at
`least not equipotent is unclear, particularly since it is
`supposed to bind to the Ca2+ channel protein both
`stereospecifically and with high affinity. pH is not likely
`to be a major factor (Triggle, D., private cori:ununica(cid:173)
`tion); for example, [3H]nitrendipine binding tot-tubule
`membranes increases as the pH is increased from 4 to
`6.5, but the increase then stops [12]. [3H]Verapamil
`binding also increases as pH is increased from 5 to 7.5,
`and then stops increasing [13]. In the former case, the
`titration plot indicates the existence of an ionizable
`group with a pK of 6.5, while in the latter case, the pK
`of the ionizable group is about 5.4. Although this value
`approaches the pK of an ionizable group found in the
`Ca2+ channel of barnacle fibers [14], there is little or no
`change in the inward Ca2 + current in the pH range of
`6-8 [15].
`Several explanations for the results obtained with
`nicardipine can be given. One is simply that the drug
`does not completely block the voltage-dependent Ca2 +
`channels present in barnacle fibers because dihydro(cid:173)
`pyridine receptors are not in abundance. Another ex(cid:173)
`planation which comes from the recent work of Valdivia
`and Coronado [16] is that verapamil, but not the 1,4-di(cid:173)
`hydropyridines, is able to block the sarcoplasmic re(cid:173)
`ticulum (SR) Ca2+ release channel in skeletal muscle. If
`this be true in barnacle fibers, one would then expect
`the residual response to 100 mM K; in the presence of
`
`209
`
`nicardipine to be due to a fall in myoplasmic pCa
`resulting from the inability of nicardipine to clamp the
`SR Ca 2 + release channel. Whether the residual response
`can be abolished by the sudden removal of external
`Ca2 + or injection of EGTA remains to be seen.
`
`Special thanks are due to Dr. R. Simmons of Syntex
`Research Inc., Palo Alto, CA for a supply of nicardipine
`HCI.
`
`References
`
`1 Venter, J.C. and Triggle, D. (eds.) (1987) Structure and Physiolugy
`of the Slow Inward Calcium Channel, Alan R. Liss, New York.
`2 Borsotto, M., Barhanin, J., Fosset, M. and Lazdunski, M. (1985) J.
`Bioi. Chern. 260, 14255.
`3 Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi,
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`(1987) Nature 328, 313-318.
`4 Ellis, S.D., Williams, M.E., Ways, N.R., Brenner, R., Sharp, A.H.,
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`5 Catterall, W.A. (1988) Science 242, 50-61.
`6 Glossmann, H., Ferry, D.R. and Boschek, C.B. (1988) Naunyn·
`Schmiedeberg's Arch. Pharmacol. 323, 1-11.
`7 Curtis, B.M. and Catterall, W.A.
`(1984) Biochemistry 23,
`2113-2118.
`8 Hagiwara, S. (1983) Membrane Potential-Dependent Ion Channels
`in Cell Membrane, Raven Press, New York.
`9 Mason-Sharp, D. and Bittar. E.E. (1981) J. Membr. Bioi. 58,
`213-226.
`10 Bittar, E.E. (1983) Progr. Neurobiol. 20, 1-54.
`11 Dow, R.J. and Graham, D.J.M. (1986) Dr. J. Clin. Pharmacal. 22,
`1955-2025.
`.
`12 Fosset, M., Jainovich, E., Delpont, E. and Lazdunski, M. (1983) J.
`Bioi. Chern. 258, 6086-6092.
`13 Galizzi, J.-P., Fosset, M. and Lazdunski, M. (1984) Eur. J. Bio·
`chem. 144, 211-215.
`14 Murayama, K. and Lakshminarayanaiah, N. (1977) J. Membr.
`Bioi. 35, 257-283.
`15 Lakshminarayanaiah, N. (1981) New Perspectives on Calcium
`Antagonists, (G.B. Weiss, ed.), pp. 19-33, American. Physiological
`Society, Bethesda, MD.
`16 Valdivia, H.H. and Coronado, R. (loJ89) Biophys. J. 55, 207a.
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