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Ascorbate increases apparent “specific” binding of [3H]pyrilamine to glass fiber filters
Ascorbate Increases Apparent “Specific” Binding of [3H]pyrilamine to Glass Fiber Filters
PATRICIAB. WILLIAMSAND MIRIAM D. ROSENTHAL
Although filtration through glass fiber filters provides a convenient method for separating is often
bound
from
a problem.
serotonergic
acids to these
of [3H]pyrilamine, dition
filters
Thus,
appears
Key Words:
radioligand, (0.1%)
filters.
increased, the effect
rather
binding
been
to glass fiber than
of ascorbate
to be dependent
Histamine;
has
In the present
an HI-antagonist,
of ascorbate
to the filters. fiber
unbound
Ascorbate
Pyrilamine;
upon
of the ligand
shown
binding
study,
relatively
filters
was observed.
decreased,
binding
on binding the particular
Radioligand
to the filters
to decrease
high
The
ad-
of [3H]pyrilamine
of radioligands ligand
of
binding
to glass
involved.
binding
INTRODUCTION [3HlPyrilamine is a selective, high-affinity antagonist for histamine H-l receptors (Hill et al., 1977). It has been successfully used to characterize the H-l receptor in preparations from a wide variety of target organs such as brain (Weich and Martin, 1982), atria (Light and Hughes, 1984), aorta (Carman-Krzan, 19831, trachea and lungs (Carswell and Nahorski, 1982), as well as in isolated cells, e.g., human mononuclear cells (Casale et al., 1985). Many radioligand binding studies using [3Hlpyrilamine and other radioligands have been done with vacuum filtration through glass fiber filters. Filtration through glass fiber filters provides for reproducible, rapid separation of bound and unbound ligand. One problem, however, has been nonspecific binding of the ligand to the filter itself. Some ligands have been reported to exhibit apparent “specific” binding, in that a portion of the total binding to the filters is inhibited by an excess of nonradiolabeled agonist or antagonist. For example, radiolabeled opiates have been demonstrated to bind stereospecifically to glass fiber filters (Snyder et al., 1975). Recently, apparent “specific” binding to filters has also been described for [3H]8hydroxy-2-di-n-propylamino-tetralin ([3H18-OH-DPAT), a radioligand for serotonin receptors (Peroutka and Demopulos, 1986). For these reasons, studies of radioligand binding to tissues require utilization of controls that measure the amount of radioactive ligand binding to the filters in the
From the Departments of Pharmacology and Biochemistry, Eastern Virginia Medical School, Norfolk, Virginia. Received February 10,1988; accepted May 30,1988. Address reprint requests to: Patricia B. Williams, Ph.D., Department of Pharmacology, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501.
293 Journal of Pharmacological 0 1988 Elsevier
Science
Methods Publishing
20,293-298 Co.,
Inc.,
(1968) 655 Avenue
of the Americas,
New
York,
NY 10010
294
P. B. Williams and M. D. Rosenthal
absence of tissue. Factors that influence these control values are therefore of concern as confounding variables in the interpretation of the resultant data. Low concentrations of ascorbate are frequently included in physiologic buffers as an antioxidant. The inclusion of ascorbate in incubation media during radioligand binding may reduce the amount of radioligand specifically bound to the target tissue (Jones and Bylund, 1986). Similarly, Peroutka and Demopulos (1986) have demonstrated that low concentrations of ascorbate decrease binding of 13H18-OH-DPAT to glass fiber filters in a specific fashion that could be inhibited by nonradiolabeled serotonin. These studies were undertaken to determine if the incorporation of ascorbate into the buffers used to wash the filters during studies with [3Hlpyrilamine would similarly decrease the background binding of this radioligand to the filters. In contrast to earlier studies utilizing other ligands, ascorbate increases the apparent “specific” binding of [3H]pyrilamine to the filters. METHODS Assays of the binding of [3Hlpyrilamine to filters were conducted using the actual membrane binding protocol but in the absence of tissue. Nonradiolabeled pyrilamine (6.67 mM) was used to determine the extent of nonspecific binding. Most experiments used Tris-buffered saline, which contained 0.15 M NaCl plus 50 mM TrisHCI, pH 7.4 at 37°C. In some cases, a phosphate-buffered saline (PBS) was used; this buffer contained (in mM) NaCI, 137; KCI, 3; CaCl*, I; MgC12, 0.5; NaHP04, 8; KH2P04, 1.5; at pH 7.4 at 37°C. To start each experiment, [3H]pyrilamine (26 Ci/mmol, Amersham Corporation, Arlington Heights, IL) was added to tubes containing buffer with or without nonradiolabeled pyrilamine for a total volume of 0.6 ml. The tubes were vortexed and incubated for 15 min at 25°C. After the incubation, the [3Hlpyrilamine was rapidly diluted with 5.0 ml of a solution containing either buffer alone or buffer with ascorbate (0.1%) followed immediately by filtration through Whatman 934-AH glass fiber filters under vacuum. The filters were then rinsed twice with 5.0 ml of buffer or buffer with ascorbate. Filters were placed in scintillation vials containing 10.0 ml ACS (Amersham Corp., Arlington Heights, IL) and radioactivity was determined by liquid scintillation spectroscopy with 29% efficiency. All experiments were done in triplicate using filters presoaked in buffer containing nonradiolabeled pyrilamine (2 mM). Filters were presoaked with nonradiolabeled pyrilamine to minimize the amount of nonspecific 13H]pyrilamine binding by saturating nonspecific binding sites on the filters with the nonradiolabeled compound before exposure to the labeled ligand. Data is presented as mean t SEM. Significant differences were determined by means of Student’s t-test. Differences were considered significant when p < 0.05. RESULTS Binding of [3H]pyrilamine
to Filters
Preliminary experiments with vascular endothelial cells suggested a two-site model. Therefore, two concentrations of 13Hlpyrilamine, one from the low affinity
Effect of Ascorbate on [3H]pyrilamine
Binding
range (25 nM) and one from the high affinity range (10 PM) were selected to investigate the effects of ascorbate on binding of L3Hlpyrilamine to glass fiber filters. Oata from typical experiments are presented in Figure 1. The results indicate that, at both 25 nM and IO PM, [3~lpyrilamine appears to label specifically binding sites on glass fiber filters. At both concentrations, there was residual radioactivity that was not inhibited by an excess of nonradiolabeled pyrilamine (6.67 mM); this represents nonspecific binding. At 25 nM 13Hlpyrilamine, apparent “specific” binding accounted for 67.4% of the total binding to the filters; at IO yM [3~]pyrilamine; “specific“ binding accounted for 29.8% of the total binding. Thus, a considerable amount of apparent “specific” binding was observed under these conditions, which is particularly apparent at low concentrations of the ligand. Effect of Ascorbate on Binding of 13Hlpyrilamine Addition of ascorbate (0.1%) to the buffer used to dilute the [3H]pyrilamine and wash the filters resulted in a marked increase in total binding to the filter. At 25 nM PHIpyrilamine, the filter bound 0.072 pmol of the 13H]pyrilamine passed through
12-r”
--25nM
pyr--
--
1 OuM pyr--
I
EII
Tris
ES3 Tris -I- ascot-bate
FIGURE 1. Effect of ascorbate on binding of [3Hlpyrilamine to glass fiber filters. In separate experiments the effects of ascotbate at two concentrations, 25 nM and 10 PM, of pyrilamine fpyr) were determined. The purchased tritiated compound was diluted directly with buffer to provide the 25 nM [3H~pyrilamine. To obtain a pyrilamine concentration of 10 u&t in the assay buffer, 10 u&t nonradiolabeled pyrilamine was added to the 25 nM f%i]pyrilamine. The amount of radioactivity was kept constant. A total of 2.3 x 105 cpm were passed over each filter. The vertical bars represent the mean and the vertical lines the SEM.
295
296
P. 6. Williams and M. D. Rosenthal
---TRIS-
-__pBS__-
iij z? ‘G
0
buffer
alone
m
buffer
+ ascorbate
FIGURE 2. Effect of buffer on binding of [3H]pyrilamine to glass fiber filters. The concentration of [3H]pyrilamine was 25 nM for both buffers. The vertical bars represent the mean and the vertical lines the SEM.
it. In the presence of ascorbate (O.l%), total binding to the filter was 0.739 pmol, a tenfold increase. Similarly, at IO FM [3Hlpyrilamine, total binding to the filter was 29 pmol, while in the presence of ascorbate 70 pmol of the t3Hlpyrilamine bound to the filter, a 2.5-fold increase. Nonspecific binding was unchanged upon the addition of ascorbate. Therefore, the apparent “specific” binding of 13Hlpyrilamine to the filters also increased in the presence of ascorbate. Effect of Buffer
on Binding
of [3H]pyrilamine
In another series of experiments, phosphate-buffered saline (PBS) was substituted for Tris buffer in the reaction media and stopwash solution (Fig. 2). Both total and nonspecific binding of 13H]pyrilamine to the filters were similar in PBS buffer compared to Tris buffer; the differences were not statistically significant. When ascorbate was added, total binding to the filter increased significantly in both buffer systems. Ascorbate did not significantly affect the amount of nonspecific binding in either buffer. DISCUSSION
These results indicate a) that [3Hlpyrilamine binds “specifically” to glass fiber filters and can be inhibited by an excess of nonradiolabeled pyrilamine and b) that
Effect of Ascorbate on [3H]pyrilamine
Binding
ascorbate (0.1%) increases the total binding of 13Hlpyrilamine to the filters resulting in an increase in the apparent “specific” binding of [3Hlpyrilamine to the filters. Ascorbate does not increase binding in the presence of a large excess of nonradiolabeled pyrilamine. Thus, in this system, ascorbate increases the “specific” binding of [3H~pyrilamine to glass fiber filters. The finding that ascorbate can increase the binding of this radioligand to the filters is in contrast to reports for other radioligands indicating that ascorbate interferes with binding to the filter (Peroutka and Demopulos, 1986; Jones and Bylund, 1986). Since similar results were obtained with either PBS or Tris buffers, the apparent “specific” binding of 13H]pyrilamine to glass fiber filters was not a result of using the Tris buffer. The effect of ascorbate on binding of radioligands to glass fiber filters appears to depend upon the particular ligand. Ascorbate decreases binding of L3H18-OHDPAT to the filter (Peroutka and Demopulos, 1986) but increases binding of 13Hfpyrilamine, as in the present study. Thus, these results add another confounding role for ascorbate in addition to its possible effects on various membrane-bound receptors (Dunlap et al., 1979; Heikkila, 1983; Heikkila and Cabbat, 1983; Jones and Bylund, 7986). The results of this study indicate that radioligand binding studies must be interpreted cautiously when ascorbate is used as an antioxidant. It is necessary to demonstrate that ascorbate does not alter either the characteristics of the ligand-receptor interaction or ligand-filter background binding. This is particularly important when ascorbate is used to retard oxidation in some but not all of the antagonists used in competition studies (Carswell and Nahorski, 1982). For these reasons, we have stopped using ascorbate in tissue experiments with [3Hlpyrilamine. We thank ina Lee, M.D., and Janet E. fones, B.S., for their expert assistance. This study was supported in part by BRSG25092RO557~ awarded by the Biomedical Research Support Grant Program, NIH, and by a grant from the Fraternal Order of Eagles.
REFERENCES Carman-Krzan M (1983) Specific binding of L3H] mepyramine to histamine HI-receptors in vascular smooth muscle membranes. Agents Actions 13:162-166. Carswell H, Nahorski SR (1982) Distribution and characteristics of histamine H,-receptors in guinea pig airways identified by [3H]mepyramine. Fur / Pharmaco/81:301-307. Casale TB, Wescott S, Rodbard D, Kaliner M (1985) Characterization of histamine H-l receptors on human mononuclear cells. Int 1 Immunopharmacol7:639-645. Dunlap III CE, Leslie FM, Rado M, Cox BM (1979) Ascorbate destruction of opiate stereospecific
binding in guinea pig brain homogenate. Pharmacof 16:105-119.
Molec
Heikkila RE (1983) Ascorbate-induced lipid peroxidation and the binding of [3HlDihydroaipren0101. Eur j Ph~rmaco/ 93:79-85. Heikkila RE, Cabbat FS (19831 Ascorbate-induced lipid peroxidation and inhibition of f3H]Spiroperidol binding in neostriatal membrane preparations. 1. Neurochem 41:13&l-1392. Hill SJ, Young JM, Marrian DF (1977) Specific binding of 3H-mepyramine to histamine H1-receptors in intestinal smooth muscle. Nature 170:361-363. Jones SB, Bylund DB (1986) Ascorbic acid inhibition of alpha-adrenergic receptor binding. Biochem Pharmacol35:595-599.
297
298
P. B. Williams and M. D. Rosenthal Light KE, Hughes MJ (1984) Altered calcium concentration and rabbit atrial t+receptors. Agents Actions 15:509-512. Peroutka SJ, Demopulos CM (1986) 13H18-OH-DPAT “specifically” labels glass fiber filter paper. Eur/ Pharmacoi 129:799-200. Snyder S, Pasternak G, Pert C (1975) Opiate Ueceptor Mechanisms. In Handbook of Pyscho-
pharmacology, Vol. 5. Eds., LL Iverson, SD Iversen, and S Snyder. New York: Plenum Press, pp. 329-360. Weich NL, Martin jS (1982) Absence of an effect of terfenadine on guinea pig brain histamine H1receptors in vivo determined by receptor binding techniques. Arzneimi~elfo~chung 32:11671170.
PATRICIAB. WILLIAMSAND MIRIAM D. ROSENTHAL
Although filtration through glass fiber filters provides a convenient method for separating is often
bound
from
a problem.
serotonergic
acids to these
of [3H]pyrilamine, dition
filters
Thus,
appears
Key Words:
radioligand, (0.1%)
filters.
increased, the effect
rather
binding
been
to glass fiber than
of ascorbate
to be dependent
Histamine;
has
In the present
an HI-antagonist,
of ascorbate
to the filters. fiber
unbound
Ascorbate
Pyrilamine;
upon
of the ligand
shown
binding
study,
relatively
filters
was observed.
decreased,
binding
on binding the particular
Radioligand
to the filters
to decrease
high
The
ad-
of [3H]pyrilamine
of radioligands ligand
of
binding
to glass
involved.
binding
INTRODUCTION [3HlPyrilamine is a selective, high-affinity antagonist for histamine H-l receptors (Hill et al., 1977). It has been successfully used to characterize the H-l receptor in preparations from a wide variety of target organs such as brain (Weich and Martin, 1982), atria (Light and Hughes, 1984), aorta (Carman-Krzan, 19831, trachea and lungs (Carswell and Nahorski, 1982), as well as in isolated cells, e.g., human mononuclear cells (Casale et al., 1985). Many radioligand binding studies using [3Hlpyrilamine and other radioligands have been done with vacuum filtration through glass fiber filters. Filtration through glass fiber filters provides for reproducible, rapid separation of bound and unbound ligand. One problem, however, has been nonspecific binding of the ligand to the filter itself. Some ligands have been reported to exhibit apparent “specific” binding, in that a portion of the total binding to the filters is inhibited by an excess of nonradiolabeled agonist or antagonist. For example, radiolabeled opiates have been demonstrated to bind stereospecifically to glass fiber filters (Snyder et al., 1975). Recently, apparent “specific” binding to filters has also been described for [3H]8hydroxy-2-di-n-propylamino-tetralin ([3H18-OH-DPAT), a radioligand for serotonin receptors (Peroutka and Demopulos, 1986). For these reasons, studies of radioligand binding to tissues require utilization of controls that measure the amount of radioactive ligand binding to the filters in the
From the Departments of Pharmacology and Biochemistry, Eastern Virginia Medical School, Norfolk, Virginia. Received February 10,1988; accepted May 30,1988. Address reprint requests to: Patricia B. Williams, Ph.D., Department of Pharmacology, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501.
293 Journal of Pharmacological 0 1988 Elsevier
Science
Methods Publishing
20,293-298 Co.,
Inc.,
(1968) 655 Avenue
of the Americas,
New
York,
NY 10010
294
P. B. Williams and M. D. Rosenthal
absence of tissue. Factors that influence these control values are therefore of concern as confounding variables in the interpretation of the resultant data. Low concentrations of ascorbate are frequently included in physiologic buffers as an antioxidant. The inclusion of ascorbate in incubation media during radioligand binding may reduce the amount of radioligand specifically bound to the target tissue (Jones and Bylund, 1986). Similarly, Peroutka and Demopulos (1986) have demonstrated that low concentrations of ascorbate decrease binding of 13H18-OH-DPAT to glass fiber filters in a specific fashion that could be inhibited by nonradiolabeled serotonin. These studies were undertaken to determine if the incorporation of ascorbate into the buffers used to wash the filters during studies with [3Hlpyrilamine would similarly decrease the background binding of this radioligand to the filters. In contrast to earlier studies utilizing other ligands, ascorbate increases the apparent “specific” binding of [3H]pyrilamine to the filters. METHODS Assays of the binding of [3Hlpyrilamine to filters were conducted using the actual membrane binding protocol but in the absence of tissue. Nonradiolabeled pyrilamine (6.67 mM) was used to determine the extent of nonspecific binding. Most experiments used Tris-buffered saline, which contained 0.15 M NaCl plus 50 mM TrisHCI, pH 7.4 at 37°C. In some cases, a phosphate-buffered saline (PBS) was used; this buffer contained (in mM) NaCI, 137; KCI, 3; CaCl*, I; MgC12, 0.5; NaHP04, 8; KH2P04, 1.5; at pH 7.4 at 37°C. To start each experiment, [3H]pyrilamine (26 Ci/mmol, Amersham Corporation, Arlington Heights, IL) was added to tubes containing buffer with or without nonradiolabeled pyrilamine for a total volume of 0.6 ml. The tubes were vortexed and incubated for 15 min at 25°C. After the incubation, the [3Hlpyrilamine was rapidly diluted with 5.0 ml of a solution containing either buffer alone or buffer with ascorbate (0.1%) followed immediately by filtration through Whatman 934-AH glass fiber filters under vacuum. The filters were then rinsed twice with 5.0 ml of buffer or buffer with ascorbate. Filters were placed in scintillation vials containing 10.0 ml ACS (Amersham Corp., Arlington Heights, IL) and radioactivity was determined by liquid scintillation spectroscopy with 29% efficiency. All experiments were done in triplicate using filters presoaked in buffer containing nonradiolabeled pyrilamine (2 mM). Filters were presoaked with nonradiolabeled pyrilamine to minimize the amount of nonspecific 13H]pyrilamine binding by saturating nonspecific binding sites on the filters with the nonradiolabeled compound before exposure to the labeled ligand. Data is presented as mean t SEM. Significant differences were determined by means of Student’s t-test. Differences were considered significant when p < 0.05. RESULTS Binding of [3H]pyrilamine
to Filters
Preliminary experiments with vascular endothelial cells suggested a two-site model. Therefore, two concentrations of 13Hlpyrilamine, one from the low affinity
Effect of Ascorbate on [3H]pyrilamine
Binding
range (25 nM) and one from the high affinity range (10 PM) were selected to investigate the effects of ascorbate on binding of L3Hlpyrilamine to glass fiber filters. Oata from typical experiments are presented in Figure 1. The results indicate that, at both 25 nM and IO PM, [3~lpyrilamine appears to label specifically binding sites on glass fiber filters. At both concentrations, there was residual radioactivity that was not inhibited by an excess of nonradiolabeled pyrilamine (6.67 mM); this represents nonspecific binding. At 25 nM 13Hlpyrilamine, apparent “specific” binding accounted for 67.4% of the total binding to the filters; at IO yM [3~]pyrilamine; “specific“ binding accounted for 29.8% of the total binding. Thus, a considerable amount of apparent “specific” binding was observed under these conditions, which is particularly apparent at low concentrations of the ligand. Effect of Ascorbate on Binding of 13Hlpyrilamine Addition of ascorbate (0.1%) to the buffer used to dilute the [3H]pyrilamine and wash the filters resulted in a marked increase in total binding to the filter. At 25 nM PHIpyrilamine, the filter bound 0.072 pmol of the 13H]pyrilamine passed through
12-r”
--25nM
pyr--
--
1 OuM pyr--
I
EII
Tris
ES3 Tris -I- ascot-bate
FIGURE 1. Effect of ascorbate on binding of [3Hlpyrilamine to glass fiber filters. In separate experiments the effects of ascotbate at two concentrations, 25 nM and 10 PM, of pyrilamine fpyr) were determined. The purchased tritiated compound was diluted directly with buffer to provide the 25 nM [3H~pyrilamine. To obtain a pyrilamine concentration of 10 u&t in the assay buffer, 10 u&t nonradiolabeled pyrilamine was added to the 25 nM f%i]pyrilamine. The amount of radioactivity was kept constant. A total of 2.3 x 105 cpm were passed over each filter. The vertical bars represent the mean and the vertical lines the SEM.
295
296
P. 6. Williams and M. D. Rosenthal
---TRIS-
-__pBS__-
iij z? ‘G
0
buffer
alone
m
buffer
+ ascorbate
FIGURE 2. Effect of buffer on binding of [3H]pyrilamine to glass fiber filters. The concentration of [3H]pyrilamine was 25 nM for both buffers. The vertical bars represent the mean and the vertical lines the SEM.
it. In the presence of ascorbate (O.l%), total binding to the filter was 0.739 pmol, a tenfold increase. Similarly, at IO FM [3Hlpyrilamine, total binding to the filter was 29 pmol, while in the presence of ascorbate 70 pmol of the t3Hlpyrilamine bound to the filter, a 2.5-fold increase. Nonspecific binding was unchanged upon the addition of ascorbate. Therefore, the apparent “specific” binding of 13Hlpyrilamine to the filters also increased in the presence of ascorbate. Effect of Buffer
on Binding
of [3H]pyrilamine
In another series of experiments, phosphate-buffered saline (PBS) was substituted for Tris buffer in the reaction media and stopwash solution (Fig. 2). Both total and nonspecific binding of 13H]pyrilamine to the filters were similar in PBS buffer compared to Tris buffer; the differences were not statistically significant. When ascorbate was added, total binding to the filter increased significantly in both buffer systems. Ascorbate did not significantly affect the amount of nonspecific binding in either buffer. DISCUSSION
These results indicate a) that [3Hlpyrilamine binds “specifically” to glass fiber filters and can be inhibited by an excess of nonradiolabeled pyrilamine and b) that
Effect of Ascorbate on [3H]pyrilamine
Binding
ascorbate (0.1%) increases the total binding of 13Hlpyrilamine to the filters resulting in an increase in the apparent “specific” binding of [3Hlpyrilamine to the filters. Ascorbate does not increase binding in the presence of a large excess of nonradiolabeled pyrilamine. Thus, in this system, ascorbate increases the “specific” binding of [3H~pyrilamine to glass fiber filters. The finding that ascorbate can increase the binding of this radioligand to the filters is in contrast to reports for other radioligands indicating that ascorbate interferes with binding to the filter (Peroutka and Demopulos, 1986; Jones and Bylund, 1986). Since similar results were obtained with either PBS or Tris buffers, the apparent “specific” binding of 13H]pyrilamine to glass fiber filters was not a result of using the Tris buffer. The effect of ascorbate on binding of radioligands to glass fiber filters appears to depend upon the particular ligand. Ascorbate decreases binding of L3H18-OHDPAT to the filter (Peroutka and Demopulos, 1986) but increases binding of 13Hfpyrilamine, as in the present study. Thus, these results add another confounding role for ascorbate in addition to its possible effects on various membrane-bound receptors (Dunlap et al., 1979; Heikkila, 1983; Heikkila and Cabbat, 1983; Jones and Bylund, 7986). The results of this study indicate that radioligand binding studies must be interpreted cautiously when ascorbate is used as an antioxidant. It is necessary to demonstrate that ascorbate does not alter either the characteristics of the ligand-receptor interaction or ligand-filter background binding. This is particularly important when ascorbate is used to retard oxidation in some but not all of the antagonists used in competition studies (Carswell and Nahorski, 1982). For these reasons, we have stopped using ascorbate in tissue experiments with [3Hlpyrilamine. We thank ina Lee, M.D., and Janet E. fones, B.S., for their expert assistance. This study was supported in part by BRSG25092RO557~ awarded by the Biomedical Research Support Grant Program, NIH, and by a grant from the Fraternal Order of Eagles.
REFERENCES Carman-Krzan M (1983) Specific binding of L3H] mepyramine to histamine HI-receptors in vascular smooth muscle membranes. Agents Actions 13:162-166. Carswell H, Nahorski SR (1982) Distribution and characteristics of histamine H,-receptors in guinea pig airways identified by [3H]mepyramine. Fur / Pharmaco/81:301-307. Casale TB, Wescott S, Rodbard D, Kaliner M (1985) Characterization of histamine H-l receptors on human mononuclear cells. Int 1 Immunopharmacol7:639-645. Dunlap III CE, Leslie FM, Rado M, Cox BM (1979) Ascorbate destruction of opiate stereospecific
binding in guinea pig brain homogenate. Pharmacof 16:105-119.
Molec
Heikkila RE (1983) Ascorbate-induced lipid peroxidation and the binding of [3HlDihydroaipren0101. Eur j Ph~rmaco/ 93:79-85. Heikkila RE, Cabbat FS (19831 Ascorbate-induced lipid peroxidation and inhibition of f3H]Spiroperidol binding in neostriatal membrane preparations. 1. Neurochem 41:13&l-1392. Hill SJ, Young JM, Marrian DF (1977) Specific binding of 3H-mepyramine to histamine H1-receptors in intestinal smooth muscle. Nature 170:361-363. Jones SB, Bylund DB (1986) Ascorbic acid inhibition of alpha-adrenergic receptor binding. Biochem Pharmacol35:595-599.
297
298
P. B. Williams and M. D. Rosenthal Light KE, Hughes MJ (1984) Altered calcium concentration and rabbit atrial t+receptors. Agents Actions 15:509-512. Peroutka SJ, Demopulos CM (1986) 13H18-OH-DPAT “specifically” labels glass fiber filter paper. Eur/ Pharmacoi 129:799-200. Snyder S, Pasternak G, Pert C (1975) Opiate Ueceptor Mechanisms. In Handbook of Pyscho-
pharmacology, Vol. 5. Eds., LL Iverson, SD Iversen, and S Snyder. New York: Plenum Press, pp. 329-360. Weich NL, Martin jS (1982) Absence of an effect of terfenadine on guinea pig brain histamine H1receptors in vivo determined by receptor binding techniques. Arzneimi~elfo~chung 32:11671170.