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Evidence that arcaine increases the N-methyl-d-aspartate-induced cardiovascular effects into the periaqueductal gray area of anesthetized rats
Ncurowience Leller.~, Limb! 1994) } ¢,4 i~'~5 ~: 1994 Elsevier Science Ireland Ltd. All rights reserved 0304-3940/94/$ 07.00
164
NSL 10132
Evidence that arcaine increases the N-methyl-D-aspartate-induced cardiovascular effects into the periaqueductal gray area of anesthetized rats Sabatino Maione, Liberato Berrino, A n n a Pizzirusso, Juan Leyva, Luigi Stella, Francesco Rossi* Institute of Pharmacology and Toxicology, Faculty of Medicine and Surgery, H University of Naples, Via Costantinopoli 16, 80138 Naples, Italy (Received 6 August 1993; Revised version received 2t October 1993; Accepted 25 October 1993)
Key words. Arcaine; N-Methyl-D-aspartate; Cardiovascular system; Periaqueductal gray matter; Rat In the present study the influence of arcaine (0.01-1/lg/rat), an in vitro putative non-competitive antagonist of the NMDA receptors, on cardiovascular changes induced by intracerebral administration of N-methyl-o-aspartate (NMDA) (0.1 ~tg/rat) has been evaluated. Both NMDA and arcaine were microinjected into the periaqueductal gray (PAG) area of anesthetized rats. Arcaine did not decrease NMDA-induced arterial hypertension and tachycardia, but, in a dose-related manner, increased the NMDA-induced cardiovascular effects. Moreover, treatment with arcaine was not able per se to modify arterial blood pressure and heart rate basal values. Although updated in vitro reports indicate arcaine as a blocker of the NMDA receptors by an open channel mechanism, our in vivo results, at the level of the PAG area, show this not to be true. Indeed the drug may facilitate NMDA receptor activation.
Both c-glutamate and L-arcaine are mediators of the major excitatory pathways in the central nervous system. They activate an heterogeneous population of receptors classically subdivided into N-methyl-o-aspartate (NMDA) and non-NMDA [11]. The former ones have raised great interest because of their involvement in the genesis and maintenance of both physiological and pathological events such as hypoxia/ischemia, epilepsy and stroke [3,12]. Our recent study [10], in agreement with previous reports [2,8,9], has shown that specific neuronal subregions within the midbrain periaqueductal gray (PAG) area modulate cardiovascular function. This effect occurred via N M D A rather than non-NMDA receptor activation within this brainstem area [10]. It has been recently suggested that N M D A receptors can be modulated by polyamines, spermine and spermidine, as well as by glycine and M f + [14,16,21]. Nevertheless, a selective antagonist for the polyamine modulatory site is not yet available. Ifenprodil and SL 82.0715, originally proposed as antagonists, were not sufficiently selective [4,6,7,22]. Reynolds then reported that arcaine (diguanidinebutane) was a highly selective antagonist for this site [15]. However, Donevan and coworkers [5] have subsequently shown that arcaine, rather than interacting *Corresponding author. Fax: (39) (81) 5665877.
with polyamine modulatory site, decreased NMDA-induced currents by interfering with the Mg 2+ site within the N M D A receptor channel. Arcaine could be a new, useful, non-competitive, use-dependent NMDA receptor antagonist. In this study we have thus compared arcaine effects on NMDA-induced cardiovascular changes with (+)-5-methyl- 10,11-dihydro-5H-dibenzo[a,d]-cycloepten5,10-imine hydrogen maleate (MK-801), a well known non-competitive use-dependent N M D A receptor antagonist. Male Sprague-Dawley rats. weighing 250-300 g, were housed at constant temperature (21 + I°CI and relative humidity (60%), under a regular light/dark schedule (light 07.00-19.00 h). Food and water were freely available. On the day of the experiment, each animal was anesthetized with ethyl urethane (1.2 g/kg, i.p.). We chose this anesthetic because its effects last for at least 2-3 h. Catheters were placed in a femoral artery and vein. The femoral artery was catheterized for direct measurement of arterial blood pressure, with a pressure gauge transducer. displayed on a Hellige polygraph. The jugular vein was catheterized for the systemic administration of heparine or saline. Immediately after surgery, each rat was placed on a homeothermic temperature control blanket (Harvard Apparatus), its head fixed into a stereotaxic frame (Kopf Instruments) for the direct intracerebral admin-
165
istration of N M D A (0.01 1/,tg/rat), Dt-2-amino-5-phosphonovaleric acid (2-APV) (1 ¢tg/rat), MK-801 (0.1 1 ,ug/ rat) and arcaine (0.01~.1/,tg/rat) into the dorso- caudal PAG area. The intracerebral microinjections were carried out with double glass micropipettes (FHC Brunswick, ME, USA) with an outside tip diameter of 40-50 mm pulled by a vertical pipette puller (David Kopf Instrumments, Tujunga, CA, USA). The coordinates of the atlas of Paxinos and Watson [13] (measured in y m from the bregma AP: -7.8: L: 0.5; V: 4.5) were used. A control volume of 100 nl of saline or the same volume of drug solution was injected over a period of 5 s. After each experiment, the stereotaxic coordinates of the injection sites were checked histologically. A volume of 200 nl of methylene blue (0.2%) was injected intracerebrally 5 rain before sacrificing the rat with a high dose of pentobarbital (200 mg/kg i.v.). The animal was perfused intra-cardially with 50 ml of PBS followed by 50 ml of a 10% formalin solution in PBS. Brain was removed and immersed into saturated formaline solution for 2 rain; the injection site was ascertained by using 2 consecutive sections (40 ym), one stained with cresyl violet to identify nuclei and the other one unstained to determine the dye diffusion. Only those rats whose microinjected site was located within the dorso-caudal PAG area were used for data computation. Drugs solutions were freshly prepared and filtered on the day of experiment. The following drugs were used: NMDA, 2-APV, arcaine, ethyl urethane (Sigma Chemical Co., St Louis, MO, USA) and MK-801 (RBI, Natick, USA). Results are expressed as TABLE 1 N M D A - I N D U C E D C A R D I O V A S C U L A R E F F E C T S IN RATS P R E T R E A T E D OR N O T W I T H DL-2-AM1NO-5-PHOSPHONOVALERIC A C I D (2-APV) Changes in systolic arterial blood pressure (AABP) ( m m H g + S.E.M.) (A) and heart rate (beats per rain, Abpm + S.E.M.) (B) in anesthetized (ethyl urethane, 1.2 g/kg i.p.) rats after microinjections of N-methyl-Daspartate ( N M D A , 0.01 1/~g/rat), or its vehicle (saline), into the dorsocaudal periaqueductal gray (PAG) area and pretreated or not. 5 min before, with DL-2-amino-5-phosphonovaleric acid (2-APV, 1 ~g/rat).
Treatment
Dose (yg/rat)
n
Saline 200 nl NMDA 0.01 NMDA 0.1 NMDA 1 N M D A + 2-APV 1
8 7 8 8 6
M a x i m u m blood pressure increase ( m m H g + S.E.) 3 _+ 0.6 13 _+ 3* 26_+ 5** 43 _+ 8** 12 _+ 4 °
Heart rate (bpm + S.E.t 358.6 385.4 408 446 365
+ 7.5 _+ l 1 + 12" _+ 16"* _+ 10"
Data are shown as the mean _+ S.E.M. (n = (~8). Significant differences are shown by asterisks (*P < 0.05: **P < 0.01 vs. saline: op < 0.01 vs. N M D A 1 /,tg/rat) and have been determined by A N O V A followed by N e w m a n Keuls tests for paired groups.
A
B
6o° ......
8°t
4O
E
zo
0
, 0.01
, 0.05 b~g/rat
01
0 O]
005
0.1
wg/rat
Fig. 1. Changes in systolic arterial blood pressure (DABP) ( m m H g + S.E.M.) (A) and heart rate (beat per rain, Abpm + S.E.M.) (B) in anesthetized (ethyl urethane, 1.2 g/kg i.p.) rats alter microinjections of N-methyl-D-aspartate ( N M D A , 0.1 ,ug/rat) into the dorso-caudal periaqueductal gray (PAG) area and pretreated or not, 5 rain before, with arcaine (ARC, 0.01 0.1 /lg/rat). Data represent the mean + S.E.M. (n = 7 10). Significant differences versus N M D A are shown by asterisks (*P < 0.05) and have been determined by ANOVA followed by Newman Keuls tests for paired groups.
mean + standard error of the mean (S.E.M.), and P < 0.05 and P < 0.01 are considered as the level of significance. Statistical analysis of the cardiovascular changes was performed by analysis of variance (ANOVA), followed by Newman Keuls test for paired groups [1,18,20]. Injection of 200 nl saline into dorso-caudal PAG area did not affect either basal arterial blood pressure or heart rate. However, N M D A injected (0.01 1 ~g/rat) into the same area significantly ( P < 0 . 0 1 ) increased arterial blood pressure and heart rate in a dose-related manner (Table I). Pretreatment with 2-APV (1 yg/rat), a selective N M D A antagonist, 5 min before, significantly prevented NMDA-induced hypertension and tachycardia (Table I). The putative N M D A antagonist, arcaine, was injected in the range of doses 0.01 0.1/~g/rat, 5 min prior to a hypertensive and tachycardic dose of N M D A (0.1/~g/rat). Surprisingly, arcaine, not only did not antagonize the NMDA-induced cardiovascular effects, but significantly increased in a dose-related manner the NMDA-induced hypertension and tachycardia (Fig. 1). As expected, MK801, a non-competitive N M D A receptor antagonist, in a dose-related manner (0.1-1 /~g/rat) significantly decreased NMDA-induced cardiovascular effects (Fig. 2). Neither arcaine nor MK-801 affected basal values of arterial blood pressure and heart rate (data not shown). Arcaine, that has been suggested to interact with the NMDA-linked channel [5], in this study did not mimic the effect of MK-801, a well-known N M D A non-competitive antagonist. Furthermore, arcaine injected into the midbrain PAG area, facilitated the NMDA-induced hypertension and tachycardia. These results are inconsistent with previous in vitro observations which showed that arcaine, similarly to Mg 2+ and Zn 2+, could block NMDA-induced effects in patch-clamped neuronal
166
A
13 6
E
20
"
7 0,1
0.5 pg/rat
1
01
05
1
pg/rat
Fig. 2. Changes in systolic arterial blood pressure (zXABP) (mmHg + S.E.M.) (A) and heart rate (beats per min, Abpm _+ S.E.M.) (B) in anesthetized (ethyl urethane, 1.2 g/kg i.p.) rats after microinjections of N-methyl-D-aspartate (NMDA, 0.1 #g/rat) into the dorso-caudal periaqueductal gray (PAG) area and pretreated or not, 5 rain before, with MK-801 (0.1-1 #g/rat). Data represent the mean + S.E.M. (n = 8 10). Significant differences versus NMDA are shown by asterisks {*P < 0.05; **P < 0.01) and have been determined by ANOVA followed by Newman-Keuls tests for paired groups.
membranes [5] as well as MK-801. Such a discrepancy is difficult to explain; however, it could be suggested that in our in vivo model arcaine interacted with Mg 2+ site promoting a partial removal of the tonic Mg > block on NMDA receptors [17]. Alternatively, a displacement of a hypothetical endogenous non-competitive inhibitor of polyamine site [19] could be proposed. In conclusion, more studies are necessary to clarify the mechanism of action of arcaine on the NMDA receptors. This drug does not seem to be a step beyond ifenprodil and its more lipophylic analogue SL 82.07t5 putative antagonist on polyamine recognition site to NMDA receptors. In fact, the other two compounds are not selective since they also interacted with sigma receptors, ~t-adrenergic receptors and with C a 2+ movements [4,6,7,22]. However, arcaine showed opposite effects, both mediated by NMDA receptors, when in vitro or in vivo models were used. Financial support from MURST and CNR, Italy, is gratefully acknowledged.
8
9
10
I1
12 13 14
15
16
17
18 19
1 Burn, J.H., Finney, D.J. and Goodwin L.G., Biological Standardization, 2nd ed., Oxford Medical Press, Oxford, 1952. 2 Carrive, P. and Bandler, R., Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal gray: a correlative functional and anatomical study, Brain Res.. 541 (1991) 206-215. 3 Choi, D.W., Glutamate neurotoxicity in diseases of the nervous system, Neuron, 1 (1988) 623-634. 4 Contreras, P.C., Bremer, M.E. and Gray, N.M., lfenprodil and SL. 82.0915 potently binding of [3H]-(+)-3-PPP to sigma binding sites in rat brain, Neurosci. Lett., 116 (1990) 190-193. 5 Donevan, S.D., Jones, S.M. and Rogawsli, M.A., Areaine blocks N-methyl-D-aspartate receptor responses by an open channel mech-
20
21
22
anism: whole-cell and single-channel recording studies in cultured hippocampal neurones, Mol. Pharmacol., 41 (1992) 727 735. Honda, H., Sakai, Y., lwata. T., Ohba, S.. Kanezuka, T. and Irmo. O., EffEcts of ifenprodil tartrate on ~-adrenoreccptors and ( a movement in isolated canine saphenous veins, ,\rch. Int. Pharmacodyn.,292(1988) 112 121. Karbon, E.W., Patch, R.J., Pontecorvo, M.J. and Ferkany, ,!,W.. Ifenprodil potently interacts with [' H]-(+)-3-PPP-Ia beled sigma sites ill guinea pig brain membranes, Eur. J. Pharmacol., 176 (1990) 247 248. Keay, K.A., Depaulis, A., Breakspear, M.J. and Bandler, R., Preand subendothelial periaqueductal gray of the rat mediates different defense responses associated with hypertension, Neurosci. Soc. Abstr.. 16 {1990) 598. Lovick, T.A,, Inhibitory modulation of the cardiovascular defence response by the ventrolateraI periaqueductal gray matter in rats, Exp. Brain Res., 89 (1992) 133 139. Maione, S., Berrino, L., Vitagliano, S., Leyva, J. and Rossi, F., Interactive rote of L-glutamate and vasopressin, at the level of the PAG area. for cardiovascular tone and stereotyped behaviour, Brain Res., 597 (1992) 166 169. Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acids receptors: their classes, pharmacology and distinct properties in the function of the central nervous system, Annu. Re~, Pharmacol. Toxicol., 29 (1989) 365 402. Nicoli, R.A., Kauer, J.A. and Malenka, R.C., Tile current excitement about long-term potentiation, Neuron, 1 (1988) 97-103. Paxinos, G. and Watson, C., The rat brain in the stereotaxic coordinates, 2nd edn., Academic Press, San Diego, CA, 1986. Ramson, R.W. and Stek, N.L., Cooperative modulation of [3H]MK-801 binding to the N-methyt-r)-aspartate receptor-ion channel complex by L-glutamate, glycine and polyamines, J. Neurochem., 51 (1988) 830 836. Reynolds, l.J., Arcaine is a competitive antagonist of the polyamine site on the NMDA receptor, Eur. J. Pharmacot, t77 (1990) 215 216, Rock, D.M. and MacDonald, R.L., Spermine and related polyamines produce a voltage-dependent reduction of N-methyl-D-aspartate receptor single-channel conductance, Mol. Pharmacol., 42 (1992) 157-164. Shiekhattar, R. and Aston-Jones, G., NMDA-receptor-mediated sensory responses of brain noradrenergic neurons are suppressed by in vivo concentrations of extracellular magnesium, Synapse, 10 (1992) 103 109. Snedecor, G. and Cochran, W., Statistical methods, 6th edn., The Iowa State University Press, Ames, IA, 1978. Subramaniam, S. and McGonigle, P., Regional heterogeneity of polyamine effects on the N-methyl-D-aspartate receptor in rat brain. J. Neurochem., 60 (1993) 2276 2284. Tallarida, R.J. and Murray, R.B., Manual of Pharmacologic Calculations with Computer Programs, 2nd edn., Heidelberg, SpringerVerlag, 1987. Williams, K., Romano, C. and Molinoff, P.B., Effects of polyamines on the binding of [3H]MK-801 to the N-methyM)-aspartate receptor: pharmacological evidence for the existence of a polyamine recognition site, Mol. Pharmacol., 36 (1989) 575 581. Woodward, J.J. and Harms, J., The putative polyamine antagonists ifenprodil and SL 82.0715 enhance dopamine effiux from rat striatal slices independent of NMDA receptor activation, Eur. J. Pharmacol., 210 (1992) 265-270.
164
NSL 10132
Evidence that arcaine increases the N-methyl-D-aspartate-induced cardiovascular effects into the periaqueductal gray area of anesthetized rats Sabatino Maione, Liberato Berrino, A n n a Pizzirusso, Juan Leyva, Luigi Stella, Francesco Rossi* Institute of Pharmacology and Toxicology, Faculty of Medicine and Surgery, H University of Naples, Via Costantinopoli 16, 80138 Naples, Italy (Received 6 August 1993; Revised version received 2t October 1993; Accepted 25 October 1993)
Key words. Arcaine; N-Methyl-D-aspartate; Cardiovascular system; Periaqueductal gray matter; Rat In the present study the influence of arcaine (0.01-1/lg/rat), an in vitro putative non-competitive antagonist of the NMDA receptors, on cardiovascular changes induced by intracerebral administration of N-methyl-o-aspartate (NMDA) (0.1 ~tg/rat) has been evaluated. Both NMDA and arcaine were microinjected into the periaqueductal gray (PAG) area of anesthetized rats. Arcaine did not decrease NMDA-induced arterial hypertension and tachycardia, but, in a dose-related manner, increased the NMDA-induced cardiovascular effects. Moreover, treatment with arcaine was not able per se to modify arterial blood pressure and heart rate basal values. Although updated in vitro reports indicate arcaine as a blocker of the NMDA receptors by an open channel mechanism, our in vivo results, at the level of the PAG area, show this not to be true. Indeed the drug may facilitate NMDA receptor activation.
Both c-glutamate and L-arcaine are mediators of the major excitatory pathways in the central nervous system. They activate an heterogeneous population of receptors classically subdivided into N-methyl-o-aspartate (NMDA) and non-NMDA [11]. The former ones have raised great interest because of their involvement in the genesis and maintenance of both physiological and pathological events such as hypoxia/ischemia, epilepsy and stroke [3,12]. Our recent study [10], in agreement with previous reports [2,8,9], has shown that specific neuronal subregions within the midbrain periaqueductal gray (PAG) area modulate cardiovascular function. This effect occurred via N M D A rather than non-NMDA receptor activation within this brainstem area [10]. It has been recently suggested that N M D A receptors can be modulated by polyamines, spermine and spermidine, as well as by glycine and M f + [14,16,21]. Nevertheless, a selective antagonist for the polyamine modulatory site is not yet available. Ifenprodil and SL 82.0715, originally proposed as antagonists, were not sufficiently selective [4,6,7,22]. Reynolds then reported that arcaine (diguanidinebutane) was a highly selective antagonist for this site [15]. However, Donevan and coworkers [5] have subsequently shown that arcaine, rather than interacting *Corresponding author. Fax: (39) (81) 5665877.
with polyamine modulatory site, decreased NMDA-induced currents by interfering with the Mg 2+ site within the N M D A receptor channel. Arcaine could be a new, useful, non-competitive, use-dependent NMDA receptor antagonist. In this study we have thus compared arcaine effects on NMDA-induced cardiovascular changes with (+)-5-methyl- 10,11-dihydro-5H-dibenzo[a,d]-cycloepten5,10-imine hydrogen maleate (MK-801), a well known non-competitive use-dependent N M D A receptor antagonist. Male Sprague-Dawley rats. weighing 250-300 g, were housed at constant temperature (21 + I°CI and relative humidity (60%), under a regular light/dark schedule (light 07.00-19.00 h). Food and water were freely available. On the day of the experiment, each animal was anesthetized with ethyl urethane (1.2 g/kg, i.p.). We chose this anesthetic because its effects last for at least 2-3 h. Catheters were placed in a femoral artery and vein. The femoral artery was catheterized for direct measurement of arterial blood pressure, with a pressure gauge transducer. displayed on a Hellige polygraph. The jugular vein was catheterized for the systemic administration of heparine or saline. Immediately after surgery, each rat was placed on a homeothermic temperature control blanket (Harvard Apparatus), its head fixed into a stereotaxic frame (Kopf Instruments) for the direct intracerebral admin-
165
istration of N M D A (0.01 1/,tg/rat), Dt-2-amino-5-phosphonovaleric acid (2-APV) (1 ¢tg/rat), MK-801 (0.1 1 ,ug/ rat) and arcaine (0.01~.1/,tg/rat) into the dorso- caudal PAG area. The intracerebral microinjections were carried out with double glass micropipettes (FHC Brunswick, ME, USA) with an outside tip diameter of 40-50 mm pulled by a vertical pipette puller (David Kopf Instrumments, Tujunga, CA, USA). The coordinates of the atlas of Paxinos and Watson [13] (measured in y m from the bregma AP: -7.8: L: 0.5; V: 4.5) were used. A control volume of 100 nl of saline or the same volume of drug solution was injected over a period of 5 s. After each experiment, the stereotaxic coordinates of the injection sites were checked histologically. A volume of 200 nl of methylene blue (0.2%) was injected intracerebrally 5 rain before sacrificing the rat with a high dose of pentobarbital (200 mg/kg i.v.). The animal was perfused intra-cardially with 50 ml of PBS followed by 50 ml of a 10% formalin solution in PBS. Brain was removed and immersed into saturated formaline solution for 2 rain; the injection site was ascertained by using 2 consecutive sections (40 ym), one stained with cresyl violet to identify nuclei and the other one unstained to determine the dye diffusion. Only those rats whose microinjected site was located within the dorso-caudal PAG area were used for data computation. Drugs solutions were freshly prepared and filtered on the day of experiment. The following drugs were used: NMDA, 2-APV, arcaine, ethyl urethane (Sigma Chemical Co., St Louis, MO, USA) and MK-801 (RBI, Natick, USA). Results are expressed as TABLE 1 N M D A - I N D U C E D C A R D I O V A S C U L A R E F F E C T S IN RATS P R E T R E A T E D OR N O T W I T H DL-2-AM1NO-5-PHOSPHONOVALERIC A C I D (2-APV) Changes in systolic arterial blood pressure (AABP) ( m m H g + S.E.M.) (A) and heart rate (beats per rain, Abpm + S.E.M.) (B) in anesthetized (ethyl urethane, 1.2 g/kg i.p.) rats after microinjections of N-methyl-Daspartate ( N M D A , 0.01 1/~g/rat), or its vehicle (saline), into the dorsocaudal periaqueductal gray (PAG) area and pretreated or not. 5 min before, with DL-2-amino-5-phosphonovaleric acid (2-APV, 1 ~g/rat).
Treatment
Dose (yg/rat)
n
Saline 200 nl NMDA 0.01 NMDA 0.1 NMDA 1 N M D A + 2-APV 1
8 7 8 8 6
M a x i m u m blood pressure increase ( m m H g + S.E.) 3 _+ 0.6 13 _+ 3* 26_+ 5** 43 _+ 8** 12 _+ 4 °
Heart rate (bpm + S.E.t 358.6 385.4 408 446 365
+ 7.5 _+ l 1 + 12" _+ 16"* _+ 10"
Data are shown as the mean _+ S.E.M. (n = (~8). Significant differences are shown by asterisks (*P < 0.05: **P < 0.01 vs. saline: op < 0.01 vs. N M D A 1 /,tg/rat) and have been determined by A N O V A followed by N e w m a n Keuls tests for paired groups.
A
B
6o° ......
8°t
4O
E
zo
0
, 0.01
, 0.05 b~g/rat
01
0 O]
005
0.1
wg/rat
Fig. 1. Changes in systolic arterial blood pressure (DABP) ( m m H g + S.E.M.) (A) and heart rate (beat per rain, Abpm + S.E.M.) (B) in anesthetized (ethyl urethane, 1.2 g/kg i.p.) rats alter microinjections of N-methyl-D-aspartate ( N M D A , 0.1 ,ug/rat) into the dorso-caudal periaqueductal gray (PAG) area and pretreated or not, 5 rain before, with arcaine (ARC, 0.01 0.1 /lg/rat). Data represent the mean + S.E.M. (n = 7 10). Significant differences versus N M D A are shown by asterisks (*P < 0.05) and have been determined by ANOVA followed by Newman Keuls tests for paired groups.
mean + standard error of the mean (S.E.M.), and P < 0.05 and P < 0.01 are considered as the level of significance. Statistical analysis of the cardiovascular changes was performed by analysis of variance (ANOVA), followed by Newman Keuls test for paired groups [1,18,20]. Injection of 200 nl saline into dorso-caudal PAG area did not affect either basal arterial blood pressure or heart rate. However, N M D A injected (0.01 1 ~g/rat) into the same area significantly ( P < 0 . 0 1 ) increased arterial blood pressure and heart rate in a dose-related manner (Table I). Pretreatment with 2-APV (1 yg/rat), a selective N M D A antagonist, 5 min before, significantly prevented NMDA-induced hypertension and tachycardia (Table I). The putative N M D A antagonist, arcaine, was injected in the range of doses 0.01 0.1/~g/rat, 5 min prior to a hypertensive and tachycardic dose of N M D A (0.1/~g/rat). Surprisingly, arcaine, not only did not antagonize the NMDA-induced cardiovascular effects, but significantly increased in a dose-related manner the NMDA-induced hypertension and tachycardia (Fig. 1). As expected, MK801, a non-competitive N M D A receptor antagonist, in a dose-related manner (0.1-1 /~g/rat) significantly decreased NMDA-induced cardiovascular effects (Fig. 2). Neither arcaine nor MK-801 affected basal values of arterial blood pressure and heart rate (data not shown). Arcaine, that has been suggested to interact with the NMDA-linked channel [5], in this study did not mimic the effect of MK-801, a well-known N M D A non-competitive antagonist. Furthermore, arcaine injected into the midbrain PAG area, facilitated the NMDA-induced hypertension and tachycardia. These results are inconsistent with previous in vitro observations which showed that arcaine, similarly to Mg 2+ and Zn 2+, could block NMDA-induced effects in patch-clamped neuronal
166
A
13 6
E
20
"
7 0,1
0.5 pg/rat
1
01
05
1
pg/rat
Fig. 2. Changes in systolic arterial blood pressure (zXABP) (mmHg + S.E.M.) (A) and heart rate (beats per min, Abpm _+ S.E.M.) (B) in anesthetized (ethyl urethane, 1.2 g/kg i.p.) rats after microinjections of N-methyl-D-aspartate (NMDA, 0.1 #g/rat) into the dorso-caudal periaqueductal gray (PAG) area and pretreated or not, 5 rain before, with MK-801 (0.1-1 #g/rat). Data represent the mean + S.E.M. (n = 8 10). Significant differences versus NMDA are shown by asterisks {*P < 0.05; **P < 0.01) and have been determined by ANOVA followed by Newman-Keuls tests for paired groups.
membranes [5] as well as MK-801. Such a discrepancy is difficult to explain; however, it could be suggested that in our in vivo model arcaine interacted with Mg 2+ site promoting a partial removal of the tonic Mg > block on NMDA receptors [17]. Alternatively, a displacement of a hypothetical endogenous non-competitive inhibitor of polyamine site [19] could be proposed. In conclusion, more studies are necessary to clarify the mechanism of action of arcaine on the NMDA receptors. This drug does not seem to be a step beyond ifenprodil and its more lipophylic analogue SL 82.07t5 putative antagonist on polyamine recognition site to NMDA receptors. In fact, the other two compounds are not selective since they also interacted with sigma receptors, ~t-adrenergic receptors and with C a 2+ movements [4,6,7,22]. However, arcaine showed opposite effects, both mediated by NMDA receptors, when in vitro or in vivo models were used. Financial support from MURST and CNR, Italy, is gratefully acknowledged.
8
9
10
I1
12 13 14
15
16
17
18 19
1 Burn, J.H., Finney, D.J. and Goodwin L.G., Biological Standardization, 2nd ed., Oxford Medical Press, Oxford, 1952. 2 Carrive, P. and Bandler, R., Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal gray: a correlative functional and anatomical study, Brain Res.. 541 (1991) 206-215. 3 Choi, D.W., Glutamate neurotoxicity in diseases of the nervous system, Neuron, 1 (1988) 623-634. 4 Contreras, P.C., Bremer, M.E. and Gray, N.M., lfenprodil and SL. 82.0915 potently binding of [3H]-(+)-3-PPP to sigma binding sites in rat brain, Neurosci. Lett., 116 (1990) 190-193. 5 Donevan, S.D., Jones, S.M. and Rogawsli, M.A., Areaine blocks N-methyl-D-aspartate receptor responses by an open channel mech-
20
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