Peripheral versus central potencies of N-type voltage-sensitive calcium channel blockers
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Naunyn-Schmiedeberg’s Arch Pharmacol (1998) 357 : 159–168
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© Springer-Verlag 1998
O R I G I N A L A RT I C L E
Y.-X. Wang · S. Bezprozvannaya · S. S. Bowersox · L. Nadasdi · G. Miljanich · G. Mezo · D. Silva · K. Tarczy-Hornoch · R. R. Luther
Peripheral versus central potencies of N-type voltage-sensitive calcium channel blockers
Received: 12 June 1997 / Accepted: 17 October 1997
Abstract The ability of a series of synthetic analogues of ω-conopeptides MVIIA (SNX-111) and TVIA (SNX-185) to prevent electrically-evoked norepinephrine release from rat tail artery and hippocampal slice preparations was determined in an effort to identify voltage-sensitive calcium channel (VSCC) blockers that selectively target N-type VSCCs in central nervous system tissue. Electrical field stimulation (3 Hz, 1 ms in duration, 80 V for 1 min) caused a high and consistent tritium outflow from rat tail artery and hippocampal slice preparations preloaded with [3H]-norepinephrine. All conopeptides, chosen for their selective affinities for high-affinity SNX-111 binding sites (i.e., N-type VSCCs) over high-affinity ω-conopeptides MVIIC (SNX-230) binding sites (i.e., P/Q-type VSCCs), produced a concentration-dependent inhibition of calcium dependent electrically-evoked tritium outflow from both tail arteries and hippocampal slices; IC50s ranged from 1.2 nM to 1.2 µM. Blocking potencies (IC50s) in the tail artery assay were significantly correlated with those measured in the hippocampal slice preparation (r = 0.91, P = 0.00000012). There was a significant correlation between IC50s for blockade of hippocampal norepinephrine release and the inhibition of high-affinity [125I]-SNX-111 binding in rat brain synaptosomes (r = 0.76, P = 0.00028). Blockade of hippocampal norepinephrine release was not significantly correlated with the inhibition of high-affinity SNX-230 binding (r = 0.46, P = 0.056). Maximum inhibition of tritium outflow in the tail artery assay was 22 ± 1.4% of control, approximating the value (20.9 ± 16.0% of control) obtained in the absence of extracellular Ca2+.
Y.-X. Wang (Y) · S. S. Bowersox · R.R. Luther Departments of Pharmacology, Neurex Corporation, 3760 Haven Avenue, Menlo Park, CA 94025, USA S. Bezprozvannaya · G. Miljanich · K. Tarczy-Hornoch Department of Biochemistry, Neurex Corporation, 3760 Haven Avenue, Menlo Park, CA 94025, USA L. Nadasdi · G. Mezo · D. Silva Department of Synthetic Chemistry, Neurex Corporation, 3760 Haven Avenue, Menlo Park, CA 94025, USA
In contrast, the maximum inhibition of tritium release from hippocampal slices was 36.8 ± 2.5% of control (P < 0.05, compared to that of the tail artery assay). These results suggest that (1) N-type VSCCs alone mediate low frequency electrical stimulation-evoked neurotransmitter release from peripheral sympathetic efferents (tail artery) while both N-type and non-N type(s) mediate neurotransmitter release from CNS neurons (hippocampus); and (2) analogues of ω-conopeptides MVIIA and TVIA do not differentiate between N-type VSCCs mediating norepinephrine release from central and peripheral neural tissues. Key words Calcium channels · ω-conotoxin · Conopeptide · N-type calcium channels · Transmitter release · SNX-111 · Norepinephrine
Introduction Multiple sub-types of voltage-sensitive calcium channels (VSCCs), such as L-, N-, T-, P, Q-, and O-type, have been identified in neuronal tissues, primarily based on their distinct molecular, electrophysiological, and pharmacological properties (see reviews by Tsien et al. 1991; Olivera et al. 1994; Miljanich and Ramachandran 1995). VSCCs are multisubunit proteins in which the particular isoform or subtype is determined largely by the α1 pore-forming subunit isoform. For example, P and Q VSCC subtypes contain the α1 A subunit isoform and the N VSCC subtype contains the α1 B. The interaction of ω-conotoxins with non-L-type VSCCs in brain synaptosomal preparations has been investigated with the aid of well-characterized [125I] and [127I] derivatives of the tyrosine in these peptides (see review by Miljanich and Ramachandran 1995). Binding studies clearly show that ω-conotoxin GVIA binds to a high-affinity binding sites that correspond to Ntype VSCCs (Olivera et al. 1987; McCleskey et al. 1987; Feldman et al. 1987; Plummer et al. 1989; Carbone et al. 1990; Kristipati et al. 1994). ω-Conotoxin MVIIC, or its synthetic equivalent SNX-230, selectively binds to high-
affinity sites distinct from high-affinity ω-conotoxin GVIA binding sites (Hillyard et al. 1992; Kristipati et al. 1994) that correspond to P/Q VSCCs (Randall et al. 1993; Zhang et al. 1993; Sather et al. 1993; Bowersox et al. 1995). SNX-111, a synthetic version of naturally occurring ωconotoxin MVIIA, is a selective and reversible blocker of N-type VSCCs. Binding and competition studies show that this conopeptide acts at the same binding sites as ωconotoxin GVIA with extremely high potency (Olivera et al. 1987; Kristipati et al. 1994) and has low affinity for SNX-230 binding sites in brain synaptosomes (Kristipati et al. 1994). In addition, SNX-111 inhibits calcium influx through N-type but not L-type VSCCs in IMR-32 human neuroblastoma cells, and does not inhibit, up to 10 µM, calcium influx in rat GH3 anterior pituitary cells expressing L- and T-type VSCCs (Tarczy-Hornoch et al., unpublished data), or L-type calcium current in rat sympathetic ganglion neurons (Rock et al., unpublished data). SNX111 is currently under clinical investigation for the treatment of chronic pain and head trauma. SNX-185, a synthetic version of ω-conotoxin TVIA, has a VSCC subtype selectivity similar to SNX-111 (Bowersox et al., 1992; Tarczy-Hornoch et al., unpublished data). N-type VSCCs are found in presynaptic nerve terminals where they permit the calcium influx necessary for depolarization-induced transmitter release from both central and peripheral nerves (see review by Olivera et al. 1994). Although the role N-type VSCCs play in regulating transmitter release has been extensively studied, mainly via applications of ω-conotoxin GVIA and ω-conotoxin MVIIA or SNX-111, it has not been proven that N-type VSCCs in the peripheral nervous system (PNS) are functionally identical to those expressed in the central nervous system (CNS) (see reviews by Olivera et al. 1994; Miljanich and Ramachandran 1995). Because molecular genetic studies have revealed a large potential for combinatorial structural and functional heterogeneity of VSCCs, it is possible that splice variants of the N-type α1 B subunit and/or heterogeneous combinations of VSCC subunits yield functional differences between N-type VSCCs in central and peripheral nerves. Indeed, it has been recently reported that distinct variants of the N-type VSCC α1 B subunit are differentially expressed in rat brain and sympathetic neurons and that the dominant N-type VSCC isoform in sympathetic ganglia has different gating kinetics and activates at more positive voltages than the isoform that dominates in the brain (Lin et al. 1997). This finding has important clinical implications, as SNX-111 has been shown to be a potent neuroprotective agent in rats when administered either intracerebroventricularly or intravenously (Smith and Siesjo 1992; Valentino et al. 1993; Buchan et al. 1994; Yamada et al. 1994; Zhao et al. 1994; Takizawa et al. 1995), and its neuroprotective properties have been suggested to be due to inhibition of excessive release of neurotransmitters during ischemic insult (Yamada et al. 1994; Takizawa et al. 1995). When administered systemically, SNX-111 or ω-conotoxin GVIA causes hypotension in rats, rabbits and orthostatic hypotension in normal human subjects which is mediated, in part, by the blockade of N-type
VSCCs in sympathetic efferents (Preneau and Angus 1990; Shapira et al. 1990; Bowersox et al. 1992; Luther et al. 1995; Hawkes et al. 1995; McGuire et al. 1997). The identification of compounds that differentiate between central and peripheral forms of the N-type VSCC might therefore prove useful in the development of non-sympatholytic calcium channel blockers for the treatment of ischemic brain injury. In an effort to identify N-type VSCC blockers with increased selectivity for the CNS over the PNS N-type VSCCs, we selected eighteen analogues of SNX-111 and SNX-185, based on their relative binding affinities for high-affinity SNX-111 binding sites over high-affinity SNX-230 binding sites, to determine their effects on electrically-evoked norepinephrine release from in vitro rat tail artery and hippocampal slice preparations. Tail arteries and hippocampi were chosen because they probably best reflect our primary interest in correlating the hypotensive effects of the omega-conopeptides with their ability to protect hippocampal neurons from ischemia-induced degeneration. The effects of these conopeptides on norepinephrine release were also compared with those on electrically evoked pressor responses in pithed rats. Preliminary results of this investigation were reported previously in abstract form (Wang et al. 1995).
Methods Animals. All experiments involving live animals were carried out in accordance with accepted principles of laboratory animal care and use as set forth by the National Institutes of Health. Male Sprague-Dawley rats (Simonsen Laboratories, Gilroy, Calif., USA) were used. All animals were acclimatized to the laboratory environment for a least 1 week before entering the study. Test compounds. Amino acid sequences of 18 synthetic ω-conopeptides used in this study are listed in Table 1. SNX-111 and SNX-185 are equivalent to ω-conopeptide MVIIA originally isolated from Conus magus and ω-conopeptide TVIA originally isolated from Conus tulipa (see review by Miljanich and Ramachandran 1995), respectively. All peptides were prepared as trifluoroacetate or acetate salts by solid phase synthesis following procedures described by Nadasdi et al. (1995). All the conopeptides were dissolved and diluted in saline. Tetrodotoxin, atropine sulfate, gallamine triethiodide were obtained from Sigma Chemical Company (St. Louis, Mo., USA) and were dissolved in saline. Radioligand binding assays. Binding to high-affinity ω-conopeptide MVIIA (SNX-111) and MVIIC (SNX-230) binding sites in rat brain synaptosomes was determined by measuring IC50 values of test compounds in competitive radioligand binding assays. Monoiodo radioiodinated SNX-111 and SNX-230 were prepared by the Iodogen method using Na[125I] and were purified by reverse-phase HPLC as described by Ahmad and Miljanich (1988). Binding to rat brain synaptosomes was measured as described previously (Cruz and Olivera 1986) with slight modifications (Kristipati et al. 1994). Briefly, gradient-purified rat brain synaptosomes were frozen and thawed, then diluted in ice-cold water containing a standard mixture of protease inhibitors (1 mM EGTA, 1 mM EDTA, 1 µM pepstatin, 2 µM leupeptin), homogenized with a Polytron (PT10–35, speed 6) and pelleted by centrifugation at 40,000 g for 20 min at 4° C. The supernatant was discarded and the pellet was resuspended in phosphate-buffered saline containing protease inhibitors then stored frozen until use. For binding assays, membrane suspensions were diluted to 25 µg protein/ml with binding buffer
161 Table 1 Amino acid sequences of eighteen synthetic ω-conopeptides, analogues of ω-conopeptides MVIIA and TVIA ω-Conopeptide
SNX-111b SNX-194 SNX-199 SNX-239 SNX-254 SNX-257 SNX-258 SNX-266 SNX-185c SNX-207 SNX-228 SNX-236 SNX-243 SNX-248 SNX-249 SNX-265 SNX-359 SNX-361
CKGKGAKCSRLMYDCCTGSCRSGKC [Nle12]-SNX-111 [Ala10]-SNX-111 [Leu10]-SNX-111 [Glu10]-SNX-111 [Phe13]-SNX-111 [Ala7,10]-SNX-111 [Gln10]-SNX-111 CLSXGSSCSXTSYNCCRSCNXYSRKCR [Arg10, Leu11, Met12]-SNX-185 [Leu10,11, Met12]-SNX-185 [Arg10, Leu11, Met12, Pro21]-SNX-185 [Ala6, Lys7, Arg10, Leu11, Met12]-SNX-185 [Ala3, Arg10, Leu11, Met12]-SNX-185 [Lys3, Arg10, Leu11, Met12]-SNX-185 [Met12, Pro21]-SNX-185 [Lys3]-SNX-185 [Pro21]-SNX-185
a Abbreviations for amino acids and nomenclature of peptide structure follow the recommendations of the IUPAC-IUB (1989) J Biol Chem 264: 668–673. Other abbreviations: X, hydroxyproline; Nle, norleucine. The six cysteine residues are linked as three disulfide bonds; the first cysteine to the fourth, the second to the fifth, and the third to the sixth. b represents synthetic version of ω-conopeptide MVIIA. c represents synthetic version of ω-conopeptide TVIA
(20 mM Hepes, pH 7.2, 75 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 2.0 µM leupeptin, 0.5 U aprotonin, 0.1% BSA). Stock radioiodinated peptide was diluted to approximately 300,000 cpm/ ml with binding buffer. Several dilutions of competing ligand were prepared in binding buffer. The binding assay was initiated by combining, for each data point, diluted membrane suspension, binding buffer, competing ligand solution, and diluted radioiodinated peptide into polyethylene tubes. The mixture was incubated at room temperature (22° C) for 1 h then chilled in ice water. The contents of each tube were then rapidly vacuum-filtered through a GF/C glass fiber filter that had been previously soaked in 0.6% polyethyleneimine. Filters were rinsed with 3 × 3 ml of washing buffer (20 mM Hepes, pH 7.2, 125 mM NaCl, 0.1% BSA) and counted in a gamma counter (Beckman Instruments, Palo Alto, Calif., USA). Triplicate tubes were used for each concentration of competing ligand. Tail artery norepinephrine release assay. The methods used in this study were modified from Kurz et al. (1993). Rats (220–400 g) were killed by CO2 inhalation and then decapitated. The proximal region of the tail artery was gently removed and placed in modified Krebs’ solution containing 118 mM NaCl; 4.6 mM KCl; 2.5 mM CaCl2; 1.2 mM KH2PO4; 25 mM NaHCO3; 1.2 mM MgSO4; 0.3 mM ascorbic acid; 0.03 mM EDTA, and 10 mM glucose. The solution was adjusted to pH 7.4 and equilibrated with a 95% O2-5% CO2 mixture. After removing the connective tissue, the tail artery was cut into 12–16 pieces of approximately 2–4 mm in length and incubated for 60 min in a test tube containing 2 ml modified Krebs’ solution and 0.2 µM [3H]-norepinephrine. Tail artery pieces were washed twice with [3H]-norepinephrine-free Krebs’ solution and 3–4 pieces were placed in each slice of four chambers of a superfusion apparatus (Warner Instrument Co., Hamden, CT, USA). Tissues were superfused continuously with modified Krebs’ solution containing 10 µM cocaine at a rate of approximately 1.4 ml/minute for 90 min (the end of this period was considered t = 0 min). Superfusion continued until the conclu-
sion of the experiment. Using a Grass S44 stimulator (Grass Instruments, Quincy, MA, USA), tissues were electrically stimulated (3 Hz, 1 ms duration, 80 V for 1 min) at t = –40 and t = –20 min without collecting perfusate solution. The tissues were subsequently stimulated at t = 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 min after starting the experiment (S1 to S11). Perfusate solution was collected at successive 1, 6, and 13 min intervals, beginning at t = –1 min. This collection cycle was repeated until samples were collected from S1 through S11. The values at t = 20 min (S2) were used as pretreatment controls. Test article was superfused starting at t = 20 min and each concentration of test article was superfused for 20 min. After superfusion, the contents of each slice chamber were solubilized in 1 ml hyamine hydroxide (ICN Radiochemicals, Irvine, Calif., USA). After adding 5 ml liquid scintillation cocktail (Ecolite; ICN Radiochemicals, Irvine, Calif., USA), the tritium content of the superfusate samples and solubilized tissues was measured using a liquid scintillation counter (Beckman Instruments, Inc., Palo Alto, Calif., USA). Tritium outflow was expressed as a fractional rate (per minute) using the following formula: % Electrically-evoked tritium outflow (fractional rate per minute ) = (stimulated tritium - basal tritium)/total tritium × 100 where stimulated tritium is one sixth of the tritium collected during the 6-min stimulation period, basal tritium is tritium collected during the 1-min pre-stimulation period, total tritium is total tritium present in the tissue at the onset of the respective collection period. Hippocampal slice assay. Rat (100–140 g) were killed by CO2 inhalation and then decapitated. The brains were quickly removed, both hippocampi were excised and cut into 270 µM slices using a McIlwain Tissue Chopper (The Mickle Lab. Engineering Co. Ltd., Gomshall, Surrey, UK), then placed in a 96-well culture plate (one slice per well). Slices were incubated for 60 min with 0.1 ml modified Krebs’ solution (composition described above) containing 0.4 µM [3H]-norepinephrine. After washing, 10 slices were placed in each of four chambers of a Warner superfusion apparatus. Assay procedures were identical to those used in the tail artery assay except that (1) tissues were superfused for 60 min before starting the experiment; (2) tissues were stimulated once 20 min before starting the experiment without collecting the superfusate solution; and (3) hippocampal slices were solubilized in 0.5 ml of 5 mM EDTA in 1% SDS. Blood pressure recording in pithed rats. Each rat was deeply anesthetized with pentobarbital (65 mg/kg, i.p.) and cannulae were placed in the femoral artery and tail vein. The arterial line was attached to a pressure transducer (Electromedics, Englewood, CO, USA) for continuous recording of blood pressure; the venous line was used for compound injection. Core body temperature was monitored and maintained at 37° C throughout the experiment. After parasympathetic blockade (20 µg/kg atropine, i.v.), a tracheostomy was performed and a polyethylene (PE-200) ventilation tube was inserted into the trachea. The rat was then pithed by inserting a steel rod through the eye socket and down the spinal canal to the first sacral vertebra. Immediately afterwards, the animal was paralyzed with gallamine (20 mg/kg, i.v.) and artificial respiration was instituted using a rodent respirator (CWE Inc., Ardmore, Pa., USA). An indifferent electrode was inserted under the skin of the hindlimb and the pithing rod was used to deliver electrical stimulation (50 V, 5 Hz, 1 ms pulse width, 15 s duration) to the sympathetic outflow. Baseline blood pressure responses to spinal cord stimulation were measured 3 to 5 times in successive 10 min intervals. The animal was then given an intravenous bolus injection of vehicle or test compound. One minute later, blood pressure responses to spinal stimulation were again measured. Test compound doses were increased at 10-min intervals until a maximum inhibitory response was obtained. Data analysis. For calculating IC50 or ID50, Hill coefficient (nH), minimum effect (Emin) and maximum effect (Emax) in the tail artery
162 Table 2 Inhibition of highaffinity ω-conopeptide [125I]MVIIA ([125I]-SNX-111) and ω-conopeptide [125I]-MVIIC ([125I]-SNX-230) binding by ω-conopeptide analogues. The amount of [125I]-SNX-111 or [125I]-SNX-230 bound to rat brain synaptosomes was measured in the presence of increasing amounts of the indicated test compound as described under “Method”. IC50 values are geometric means
SNX-111 binding IC50 (M)
SNX-230 binding IC50 (M)
SNX-111 SNX-185 SNX-194 SNX-199 SNX-207 SNX-228 SNX-236 SNX-239 SNX-243 SNX-248 SNX-249 SNX-254 SNX-257 SNX-258 SNX-265 SNX-266 SNX-359 SNX-361
7.2 × 10–12 2.5 × 10–10 1.0 × 10–11 3.0 × 10–10 1.2 × 10–11 3.0 × 10–10 2.9 × 10–11 2.6 × 10–10 3.4 × 10–12 1.2 × 10–11 3.1 × 10–11 7.4 × 10–9 2.0 × 10–9 1.8 × 10–8 1.1 × 10–10 2.7 × 10–10 1.1 × 10–10 6.6 × 10–9
115 11 8 2 6 1 1 1 1 1 3 1 1 2 3 1 1 1
3.9 × 10–7 7.1 × 10–6 1.2 × 10–6 1.0 × 10–5 5.2 × 10–7 8.6 × 10–6 1.5 × 10–6 9.6 × 10–6 2.6 × 10–7 1.5 × 10–6 4.5 × 10–9 5.4 × 10–6 5.7 × 10–6 1.1 × 10–5 1.7 × 10–6 1.0 × 10–5 4.4 × 10–6 1.8 × 10–6
13 11 2 1 1 1 1 1 1 2 2 2 1 2 1 1 1 1
assay, hippocampal slice assay, and the blood pressure assay in pithed rats, a four parameter logistic concentration-response curve was fitted by a Hewlett-Packard computer using a nonlinear least square method to data pooled from several experiments/samples. The formula was as follows: Effect = Emin + (Emax – Emin)/(1 + (IC50/C)nH) where Emin = effect at 0 concentration of test article, Emax = effect at infinite concentration of test article, C = concentration of test article, IC50 = concentration of test article producing 50% inhibition, and nH = Hill coefficient or slope. IC50s were also calculated using the above formula for the binding data from each experiment. Geometric means were calculated from different binding experiments, due to the empirical observation that the distribution of the log IC50 is very close to a normal distribution (Draper and Smith 1981). Incidentally, the distribution of the non-fitted data (such as electrically-evoked tritium overflow and% control used in the present study) generally follows a normal distribution, therefore the arithmetic means ± SEM were used for these values. Data sets were compared by linear regression analysis and by the two-tailed Student’s t-test.
the binding of either iodinated SNX-111 or iodinated SNX-230 to their receptors in rat brain (Kristipati et al. 1994). The affinities of each of the panel of ω-conopeptides for both N-type and P/Q-type VSCC were obtained by determining IC50s of displacement of binding of [125I]-
Results Displacement of high-affinity SNX-111 and SNX-230 binding sites in hippocampal synaptosomes Binding analysis in brain synaptosomes showed that highaffinity [125I]-SNX-111 binding sites define the N-type VSCC while high-affinity [125I]-SNX-230 binding sites define another subtype, i.e., a non-L-, non-N-type VSCC (Kristipati et al. 1994), which are probably P/Q-type VSCCs (Bowersox et al. 1995). Nonspecific binding, the binding observed in the presence of a large excess (for example, 10 nM for SNX-111) of the corresponding uniodinated conopeptide, was less than 3% of the total hot ligand, and over the concentration range tested was less than 25% of the specific binding. Moreover, the L-type VSCC blockers diltiazem, verapamil, and nimodipine, and the glutamate NMDA receptor non-competitive antagonist MK-801, at concentrations up to 100 µM, did not significantly inhibit
Fig. 1 Time dependence of electrically-evoked tritium overflow from tail artery (A) and hippocampal slice (B) preparations preloaded with [3H]-norepinephrine. Field stimulation (3 Hz, 1 ms duration and 80 V) was delivered each time to tissues for 1 min at 20 min-interval throughout the experiment. The electrically-evoked tritium overflow was calculated as% fractional rate per min as described under “Methods”. Data are means ± SEM (n = 4)
SNX-111 and [125I]-SNX-230, respectively, to rat brain synaptic membranes. SNX-111 and SNX-185 displayed roughly 50,000- and 30,000-fold, respectively, higher affinity for N-type VSCCs (SNX-111 displacement IC50s: 7.2 × 10–12 and 2.5 × 10–10 M, respectively) than for P/Qtype VSCCs (SNX-230 displacement IC50s: 3.9 × 10–7 and 7.1 × 10–6 M, respectively). As shown in Table 2, eighteen conopeptides that have about 3–5 orders of magnitude higher affinity for [125I]-111 binding sites than [125I]-230 binding sites were chosen for further studies.
Experiments were performed to determine whether electrically-evoked [3H]-norepinephrine release was dependent on nerve stimulation (Na+ channel-sensitive) or extracellular Ca2+. The sodium channel blocker, tetrodotoxin (1 µM, superfusion for 20 min), prevented electrically-evoked tritium outflow (Fig. 2 A). Superfusion with Ca2+-free modified Krebs’ solution containing 1 mM EDTA for 20 min reduced stimulation-induced responses to 21 ± 16% of control; addition of 2.5 mM Ca2+ restored the responses (Fig. 2 B). SNX-111 inhibited electrically-evoked tritium outflow from tail arteries in a concentration-dependent manner with an IC50 of 1.2 × 10–9 M and an Emax of 21% of control (Fig. 3). The time-course of SNX-111-mediated Inhibition of electrically-evoked [3H]-norepinephrine blockade of [3H]-norepinephrine release from rat tail arrelease from rat tail arteries teries was also evaluated. Superfusion of 5 nM SNX-111 Electrical stimulation increased tritium outflow from rat tail for 20 min decreased tritium outflow to 49.2 ± 6.7% of arteries preloaded with [3H]-norepinephrine. Prior to test control. The inhibitory effect gradually recovered after compound challenge, electrically-evoked tritium outflow washing with SNX-111-free media and reached 83.0 ± was 0.040 ± 0.002% (mean ± SEM, n = 112) of the total 7.4% of control values after 160 min (Fig. 4). The other 17 conopeptides tested all blocked electriradioactivity in tissue per min stimulation and the stimu3 lation/basal ratio was 9.6 ± 0.2 (mean ± SEM, n = 120). cally-evoked [ H]-norepinephrine release from rat tail arIn control experiments, the fractional rate of basal tritium teries in a concentration-dependent manner. IC50s of these outflow declined with time (data not shown); however, no compounds varied from 1.2 nM to 1.2 µM and maximum significant changes in electrically-evoked tritium outflow inhibition (Emax) ranged from 11.2 to 36.9% of control occurred throughout the sequence of stimulation, thus re- (mean ± SEM = 22.0 ± 1.4% of control) (Table 3). sulting in a consistent Sn/S2 ratio close to unity (Fig. 1 A).
Fig. 3 Concentration-dependent blockade by SNX-111 of electrically-evoked tritium outflow from rat tail arteries and hippocampal slices preloaded with [3H]-norepinephrine. Data are means ± SEM (n = 4)
Fig. 2 Blocking effects and reversibility of 1 µM tetrodotoxin (TTX) (A) and free extracellular [Ca2+] plus 1 mM EDTA (B) on electrically-evoked tritium outflow from rat tail arteries preloaded with [3H]-norepinephrine. In (A), a denotes statistically significant difference from control bar (P < 0.05); b denotes statistically significant difference from TTX (1 µM) bar. Histograms in the lower panel (B) show evoked tritium outflow in the presence of 2.5 mM Ca2+ (left), 20 min after removing Ca2+ by addition of 1 mM EDTA (center), and 20 min after adding 2.5 mM Ca2+ back to the incubation medium (right). Data are means ± SEM (n = 3 or 4). a denotes statistically significant difference from 2.5 mM Ca2+ bar (P < 0.05)
Fig. 4 Reversibility of SNX-111-induced blockade of electricallyevoked tritium outflow from rat tail arteries preloaded with [3H]norepinephrine. Data are means ± SEM (n = 4)
164 Table 3 Inhibitory effects of synthetic ω-conopeptides on electrically-evoked tritium outflow from rat tail arteries and hippocampal slices preloaded with [3H]-norepinephrine. IC50s, Hill coefficients (nHs), Emins, and Emaxs were calculated by fitting a four parameter logistic concentration-response curve as described under “Methods” to data from 1–3 experiments, each consisting of 3–4 samples. Emin was 100% for each conopeptide
SNX-111 SNX-185 SNX-194 SNX-199 SNX-207 SNX-228 SNX-236 SNX-239 SNX-243 SNX-248 SNX-249 SNX-254 SNX-257 SNX-258 SNX-265 SNX-266 SNX-359 SNX-361
Tail artery assay
Hippocampal slice assay
Emax (% control)
Emax (% control)
4 3 4 4 4 8 4 4 3 7 4 4 4 4 4 4 4 4
21.0 23.9 21.2 23.9 16.6 20.0 17.8 14.8 24.8 28.1 22.6 36.9 24.5 30.1 20.4 23.7 14.8 11.2
1.21 5.54 3.89 72.9 19.4 64.4 17.2 11.3 2.68 5.13 8.67 1 148 537 439 8.27 33.0 8.03 26.3
2.0 1.6 1.2 1.2 1.2 1.6 1.3 0.8 1.2 1.6 1.5 1.4 1.0 2.4 1.1 1.4 1.3 0.5
4 7 3 3 4 8 4 3 3 12 4 3 4 6 3 4 7 3
26.7 40.7 37.5 21.1 59.3 40.9 31.5 30.5 43.1 38.0 32.6 30.5 19.5 31.4 38.9 46.0 38.0 56.4
5.51 8.92 15.7 47.2 6.10 25.3 14.9 33.8 4.66 12.2 10.5 1 198 379 152 17.0 26.6 5.51 22.4
0.8 2.0 0.7 0.6 2.2 2.1 0.8 0.7 1.2 0.8 0.9 1.2 0.5 0.6 3.2 0.7 0.9 2.2
Inhibition of electrically-evoked [3H]-norepinephrine release from hippocampal slices Compared to the tail artery assay, electrical stimulation of hippocampal slices in the absence of test article challenge caused a much higher increase in tritium outflow; at S2, electrically-evoked tritium outflow was 0.54 ± 0.02% (mean ± SEM, n = 59) of the total radioactivity in tissue per min of stimulation and the stimulation/basal ratio was 15.6 ± 0.4 (mean ± SEM, n = 59). As in the tail artery assay, electrically-evoked tritium outflow within each experiment (Fig. 1 B) was consistent between trials (i.e., 20% rundown). Electrically-evoked [3H]-norepinephrine release from hippocampal slices was also Ca2+-dependent (data not shown). As was found in the rat tail artery assay, SNX-111 (Fig. 3) and other ω-conopeptides tested blocked electricallyevoked tritium outflow in hippocampal slices in a concentration-dependent fashion. IC50 and Emax values for each compound are listed in Table 3. Emax values ranged from 19.5% to 59.3% of control (mean ± SEM = 36.8 ± 2.5% of control) and were significantly higher than those obtained in the tail artery assay (P < 0.05). We examined the relationship between blockade of [3H]-norepinephrine release from hippocampal slices and binding affinities for high-affinity SNX-111 and SNX-230 binding sites in rat brain synaptosomes given in Table 2. As shown in Fig. 5 A, there was a significant correlation between IC50s for blockade of hippocampal norepinephrine release and for inhibition of high-affinity SNX-111 binding to synaptosomal preparations (r = 0.76, P = 0.00028). The correlation between IC50s for blockade of hippocampal norepinephrine release and inhibition of high-affinity SNX-230 binding to rat brain synaptosomes was not statistically significant (r = 0.46, P = 0.056) (Fig. 5 B).
Fig. 5 Correlation between IC50 values for blockade of [3H]-norepinephrine release from hippocampal slices and for displacement of high-affinity [125I]-SNX-111 binding (A) or [125I]-SNX-230 binding (B) to rat brain synaptosomes. Hippocampal slice and binding data are from Tables 2 and 3, respectively. The line in (A) is linear regression line
IC50 values obtained in the rat tail artery assay were compared with those from the hippocampal slice assay. Fig. 6 shows that blocking potencies of ω-conopeptides in the tail artery assay were significantly correlated with those measured in the hippocampal slice assay (r = 0.91, P = 0.00000012).
165 Table 4 Inhibitory effects of synthetic ω-conopeptides administered intravenously on pressor responses to spinal cord stimulation in pithed rats
Fig. 6 Correlation between IC50 values for blockade of electrically-evoked tritium outflow from rat tail arteries and hippocampal slices preloaded with [3H]-norepinephrine. Data are from Table 3. The line is linear regression line
Inhibition of electrically-evoked pressor responses in pithed rats Effects of sixteen ω-conopeptides on electrically-evoked pressor responses were studied in pithed rats (n = 2–4 in each group). Electrical stimulation caused a pressor response in pithed rats. SNX-111 and other conopeptides were then intravenously bolus injected at doses increasingly from approximately 3.8 × 10–10 to 3.8 × 10–7 mol/kg, with an interval between doses of 20 min. SNX-111 inhibited pressor responses to spinal stimulation in a dosedependent manner, with an ID50 of 1.4 × 10–9 mol/kg and an Emax of approximately 80% inhibition (Fig. 7). SNX185, SNX-194, SNX-199, SNX-228, SNX-239, SNX-254 and SNX-266 also inhibited pressor responses. Their ID50s are listed in Table 4. The other conopeptides, namely, SNX-258, SNX-207, SNX-236, SNX-243, SNX248, SNX-249, SNX-257 and SNX-265, at the highest tested doses (3.8 × 10–7 mol/kg), did not inhibit the pressor response or caused toxic effects. To explore the relationship between ω-conopeptide-induced depressor responses in vivo and the inhibition of norepinephrine release in vitro, the blocking potencies of these eight ω-conopeptides in the tail artery assay were compared with the ability of these compounds to inhibit
ID50 (10–9 mol/kg)
SNX-111 SNX-185 SNX-194 SNX-199 SNX-207 SNX-228 SNX-236 SNX-239 SNX-243 SNX-248 SNX-249 SNX-254 SNX-257 SNX-258 SNX-266
4 3 3 3 3 3 3 3 2 2 2 3 3 3 3
1.4 28 26 1041 Not determineda 232 Not determineda 390 Not determineda Not determineda Not determineda 3129 Not determineda Not determineda 569
a ID 50 was not determined due to no inhibitory effect and/or toxic responses at the highest tested doses (approximately 4.0 × 10-7 mol/kg)
Fig. 8 Correlation between IC50s and ID50s of eight ω-conopeptides on electrically-evoked [3H]-norepinephrine release from rat tail arteries and on pressor responses to spinal cord stimulation in pithed rats. The tail artery and blood pressure data are from Tables 3 and 4, respectively. The line is linear regression line
pressor responses to spinal stimulation in the pithed rat (Table 4). IC50 and ID50 values were significantly correlated (r = 0.90, P = 0.0025) (Fig. 8).
Fig. 7 Dose-inhibitory response curve of SNX-111 administered intravenously on electrically-evoked pressor responses in pithed rats (n = 4). Data are means ± SEM
Eighteen ω-conopeptides used in this study exhibit affinities for high-affinity SNX-111 binding sites (IC50s ranging from 7.2 × 10–12 M to 1.8 × 10–8 M) and high-affinity SNX-230 binding sites (IC50s ranging from 4.5 × 10–9 M to 1.0 × 10–5 M), but have roughly 3–5 orders of magnitude higher affinity for SNX-111 binding sites than to SNX-230 binding sites, i.e., they have significantly greater selectivity for N-type than for P/Q-type VSCCs. All these conopeptides also block norepinephrine release from hippocampal slices, with IC50s ranging from 5.5 nM
to 1.2 µM. Statistical analysis shows that there is a much greater correlation between blockade of norepinephrine release from hippocampi and displacement of high-affinity SNX-111 binding sites than high-affinity SNX-230 binding sites, suggesting that these ω-conopeptides inhibit norepinephrine release from hippocampi mainly by blocking N-type VSCCs and not P/Q-type VSCCs. Blockade of L-type VSCCs is not likely to contribute to the inhibition of norepinephrine release in these studies for the following reasons. First, these conopeptides do not block L-type VSCCs. For example, SNX-111 and SNX-185 do not inhibit, at concentrations up to 10 µM, calcium influx in rat GH3 anterior pituitary cells expressing L- and T-type but not N-type VSCCs (Tarczy-Hornoch et al. unpublished data). 10 µM SNX-111 does not inhibit L-type calcium currents in rat sympathetic ganglion neurons (Rock et al., unpublished data). Second, the L-type VSCC blockers, nitrendipine and verapamil, at micromolar concentrations (saturating for L-type VSCCs) have no detectable effect on K+-stimulated norepinephrine release from hippocampal slices (Gaur et al., unpublished data). While blockade of norepinephrine release from hippocampal slice is significantly correlated with displacement of high-affinity SNX-111 binding sites for the eighteen conopeptides tested, there is a difference of approximately two to three orders of magnitude between their potencies for the two effects. The reasons for this large difference between binding affinity and potency is not completely clear, but a major factor is due to differences in the ionic compositions of the physiological solutions used for the release assays and the buffer used for the binding experiments. The binding of ω-conopeptides to calcium channels is well known to be very sensitive to the cation content of the medium (Abe et al. 1986; Kristipati et al. 1994). Divalent cations such as Ca2+ and Mg2+ at physiological concentrations are especially potent inhibitors of ω-conopeptide binding. The monovalent cation concentration in the binding buffer was approximately half that present in the physiological media. Moreover there was no added Ca2+ or Mg2+ in the binding buffer, whereas the physiological solutions contain 2.5 mM Ca2+ and 1.2 mM Mg2+. 1.0 mM Ca2+ alone can shift the appparent binding constant of SNX-111 by one order of magnitude (Kristipati et al. 1994). Ideally, we would have preferred to have compared binding affinities and functional inhibition of norepinephrine release under the same conditions. However, release assays cannot be performed in hypo-osmotic and divalent ion-free medium (see Fig. 2 B) and, despite numerous attempts by us, binding affinities could not practically be determined in physiological solutions due to the low signal and high background. Omega-conopeptide-mediated maximum inhibition of norepinephrine release from tail arteries ranged from 11.2% to 36.9% of control with an average of 22.0 ± 1.4% (mean ± SEM) of control. Electrically-evoked tritium outflows in Ca2+-free solution plus 1 mM EDTA was approximately 21%, which may represent residual tritium release attributable to intracellular Ca2+. The results indicate that the N-type VSCC is the dominant calcium channel sub-
type in peripheral efferents responsible for norepinephrine release evoked by low-frequency (such as 3 Hz used in the present study) field stimulation. This conclusion is supported by the following observations: (1) ω-conotoxin GVIA completely blocked norepinephrine release from cultured rat superior cervical ganglion cells (Hirning et al. 1988), or electrically evoked muscle contractile responses in the rat vas deferens preparation (Keith et al. 1990) and isolated small mesenteric artery (Whorlow et al. 1996); (2) the L-channel blockers, nitrendipine or felodipine, have no effect on norepinephrine release from cultured rat superior cervical ganglion cells (Hirning et al. 1988) or on contractile responses in the rat isolated small mesenteric artery (Whorlow et al. 1996). However, the above conclusion is applicable only when using low frequency stimulation patterns as in the present study. It has been reported that high frequency (10–50 Hz) stimulation produces a ωconotoxin GVIA-insensitive “residual release” of norepinephrine (recorded as excitatory junction potentials) in postganglionic sympathetic nerve terminals of the rat isolated anococcygeus muscle (Smith and Cunnane 1996a, b; 1997). In contrast to tail arteries, maximum inhibition by the same eighteen ω-conopeptides of norepinephrine release from hippocampal slices, evoked by electrical stimulation of the same frequency, ranged from 19.5% to 59.3% (mean ± SEM) of control with an average of 36.8 ± 2.5% of control; which are significantly different from those values obtained in the tail artery assay. As these conopeptides block transmitter release by blocking N-type VSCCs but not P/Q-type VSCCs, and selective Ltype VSCC blockers do not inhibit norepinephrine release from hippocampus preparations (see above), our results suggest that in contrast to tail arteries, both N-type and non-L-, non-N-type(s) (e.g., P/Q-type) VSCCs mediate norepinephrine release from hippocampal neurons. This is supported by recent publications using hippocampal preparations (Gaur et al. 1994; Fox 1994; Turner et al. 1993). Although the role N-type VSCCs play in regulating transmitter release has been extensively studied, how peripheral N-type channels compare pharmacologically to central N-type channels has not been determined (see review by Olivera et al. 1994). One of the major aims in this study was to explore whether N-type VSCCs in the CNS as represented by hippocampi, are functionally differentiable, especially in terms of transmitter release, from those expressed in the PNS as represented by tail arteries. There is a significant correlation between blockade of transmitter release from tail arteries and hippocampal slices. The results indicate that the panel of N-type VSCCs blocking conopeptides tested so far cannot distinguish central N-type VSCCs from peripheral ones. However, our findings do not suggest that a single isoform of the N-type VSCC is responsible for norepinephrine release from both central and peripheral neurons, as the failure to discriminate differences in the present study may be attributable to inadequate structural diversity of the test ligands or to the preservation of the structure of the conopeptide binding site between N-type VSCC isoforms.
In fact, distinct variants of the N-type VSCC α1 B subunit are differentially expressed in rat brain and sympathetic neurons and the dominant N-type VSCC isoform in sympathetic ganglia has different gating kinetics from the isoform that dominates in the brain (Lin et al. 1997). SNX-111 causes depressor responses in animals (Bowersox et al. 1992) and humans (Luther et al. 1995; McGuire et al. 1997). The present results demonstrate that intravenous administration of SNX-111 and SEVEN other conopeptides inhibited electrically-evoked pressor responses in pithed rats in a dose-dependent fashion, and that there is a significant correlation between blockade of electrically-evoked pressor responses in pithed rats and norepinephrine release from tail arteries. The results provide additional evidence that blockade of sympathetic transmitter release is the mechanism whereby N-type VSCC blockers produce hypotension (Bowersox et al. 1992; Hawkes et al. 1995). N-type VSCCs regulate calcium influx necessary for neurotransmitter release from autonomic efferents innervating the vasculature. As a consequence, ω-conopeptides such as SNX-111 are potent vasodilating agents and when administered systemically to humans, produce orthostatic hypotension (Luther et al. 1995; McGuire et al. 1997). In healthy individuals, recumbent blood pressure is little affected by intravenous SNX-111 administration. Patients that have sustained serious injuries and consequent blood loss have predictably high basal sympathetic outflow and, accordingly, exhibit recumbent hypotension after receiving intravenous infusions of SNX-111 (McGuire, unpublished observation). While we have established simple and effective measures for the clinical management of ω-conopeptide-induced hypotension in severely injured patients (i.e., intravascular volume replacement and alpha-adrenergic vasopressor support), understanding the mechanism of sympatholysis and its modulation by structurally divergent ω-conopeptide analogues may provide a means of developing clinically relevant centrally-acting N-type VSCC blockers that can be administered systemically without requiring pressor support to maintain blood pressure within normal limits. In summary, the results of this investigation suggest: (1) a majority of hippocampal norepinephrine release can be inhibited by blocking N-type VSCCs, (2) N-type VSCCs alone mediate transmitter release, evoked by low frequency electrical stimulation, from peripheral sympathetic efferents, whereas both N-type and non-L-, non-Ntype(s) (e.g., P/Q-type) VSCCs mediate transmitter release from CNS neurons, and (3) a panel of structurally diverse ω-conopeptides and analogues cannot discriminate between N-type VSCCs in the tail artery and those mediating norepinephrine release from hippocampal brain slices.
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