Introduction

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Clonidine has been used for decades as an effective agent in long-term antihypertensive therapy and in the acute management of hypertensive crisis. Clonidine has been demonstrated to produce a large variety of pharmacological effects due to activation of alpha-2 adrenergic receptors present in the central nervous system and other organs (Bloor, 1993). In recent years, clonidine has been increasingly used for the purpose of enhancing systemic or neuraxial anesthesia (Hayashi and Maze, 1993; Kauppila et al., 1991; Reddy et al., 1980; Spaulding et al., 1979; Sullivan et al., 1987; Yaksh, 1985). Clonidine has also been shown to be an efficacious drug to control overshoot of sympathetic activity in patients suffering from withdrawal of alcohol (Yam et al., 1992), opioids (Gold et al., 1980) or benzodiazepines. These beneficial central effects were observed after the application of high doses of intravenous clonidine (Tryba et al., 1993). Thus, clonidine is able to cross the blood-brain barrier after intravenous injection and to interact with alpha-2 adrenergic receptors present in the central nervous system. We therefore evaluated the contribution of the blood-brain barrier in controlling clonidine transfer from blood to its central nervous system receptor sites using an in vitro cell culture system consisting of primary cultures of porcine brain capillary endothelial cells.

	Methods

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Materials. Clonidine was from Fluka (Buchs, Switzerland). [³H]Aminoclonidine was from DuPont-New England Nuclear (Boston, MA). Octanol-to-100 mM phosphate buffer, pH 7.0, partition coefficients were determined after equilibration for 24 hr at room temperature using isotope-labeled tracers (Amersham, Buckinghamshire, UK, or DuPont-New England Nuclear).

Cell cultures. Primary cultures of porcine brain capillary endothelial cells were prepared as described (Audus and Borchardt, 1986) with the following modifications: Cortical gray matter from six freshly obtained porcine brains was minced and incubated in MEM (Sigma Chemical Co., St. Louis, MO) containing 0.5% dispase (Boehringer-Mannheim Biochemica, Mannheim, Germany) for 2 hr. Cerebral microvessels were obtained after centrifugation in MEM containing 13% dextran (Sigma). The microvessels were subsequently incubated in MEM containing 1 mg/ml collagenase-dispase (Boehringer-Mannheim) for 4.25 hr. The resulting cell suspension was supplemented with 10% horse serum and filtered through a 150-µm nylon mesh. Brain capillary endothelial cells were isolated on a continuous 50% Percoll gradient (Pharmacia, Uppsala, Sweden) (centrifugation at 1000 × g for 10 min). Isolated endothelial cells were filtered through a 35-µm nylon mesh before seeding with a density of 100,000 cells/cm² onto collagen/fibronectin-coated (Boehringer-Mannheim) 24-well cell culture plates (uptake assays) or a density of 200,000 cells/cm² onto polycarbonate membranes (transendothelial transport; see below). Cells were cultured under standard cell culture conditions (Audus and Borchardt, 1986) [cell culture medium: 45% MEM, 45% Ham's F-12, 100 µg/ml streptomycin, 100 µg/ml penicillin G, 100 µg/ml heparin, 13 mM NaHCO₃ and 20 mM HEPES [all from Sigma] containing 10% heat-inactivated horse serum [GIBCO BRL, Basel, Switzerland]).

Uptake assays. Uptake assays were performed at 20°C using confluent monolayers of porcine brain capillary endothelial cells at day 10. Cells were grown on 24-well cell culture plates. The surface area was 2 cm²/well. Cells were washed using transport buffer, which consisted of 122 mM NaCl, 3 mM KCl, 1.4 mM CaCl₂, 1.2 mM MgSO₄, 4 mM D-glucose, 10 mM HEPES, 25 mM NaHCO₃ and 0.4 mM K₂HPO₄, pH 7.4. Where indicated, NaCl was substituted with choline chloride, and D-glucose was adjusted to 0 or 20 mM. The reaction was initiated by the addition of 240 µl of transport buffer containing 0.3 µCi of ³H-labeled tracer of the respective substrate, sufficient unlabeled substrate to bring the medium to the desired final concentration and 0.3 µCi of the extracellular marker [¹⁴C]sucrose.

Kinetic experiments. Uptake of clonidine was measured as a function of its concentration in the incubation mixture. Incubations were performed at room temperature or 4°C. The range of concentrations used varied from 1 µM to 2 mM. For data representation and to obtain estimates of kinetic parameters, a nonlinear regression program was used (Microcal Origin version 3.5).

Transendothelial transport. For the study of transendothelial transport, up to six polycarbonate membranes (Snap-Well System, Costar, Cambridge, MA) with a confluent monolayer of porcine brain endothelial cells were mounted in a corresponding number of side-by-side diffusion cells (Costar). Both sides of the diffusion cells were filled with prewarmed transport buffer. The entire system was maintained at a constant temperature (37°C) and was supplied with 5% CO₂/95% oxygen. At time t = 0, the isotope-labeled compound to be studied was added to the donor chamber. In defined time intervals, samples were drawn from the acceptor chamber and analyzed by scintillation counting. The acceptor chamber volume was readjusted with assay buffer after each sample was taken, and counts from acceptor side samples were corrected for the amount of radioactivity removed by previous sampling. The initial rate of transport was calculated from a linear regression. P_app values were calculated according to P_app = dQ/dt·1/A/C₀, where dQ/dt is the rate of translocation, A is the surface of the polycarbonate membrane and C₀ is the initial concentration of the labeled drug (cm/min).

	Results

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Uptake vs. time. Uptake of clonidine into cultured cerebral capillary endothelial cells was measured at various time points up to 50 min (fig. 1). Propranolol was used as a reference substance. In contrast to uptake of propranolol, uptake of clonidine was nearly linear and did not reach saturation within 50 min. The finding that clonidine penetrated brain capillary endothelial cells to a lesser degree than propranolol was unexpected in view of the high hydrophobicity of clonidine. The octanol-to-100 mM phosphate buffer, pH 7.4, partition coefficients were 0.0011 ± 0.0003 (n = 3) for [¹⁴C]sucrose, 12.2091 ± 0.6802 (n = 3) for [³H]propranolol and 86.7262 ± 1.3361 (n = 5) for [³H]clonidine (values represent mean ± S.E.M. of n experiments).

View larger version (K): [in this window] [in a new window]   Fig. 1.   Time-dependent uptake of propranolol (), clonidine () and sucrose (). Symbols represent mean ± S.E.M. (vertical lines) for propranolol (5 data points), clonidine (7 data points) and sucrose (16 data points).

Kinetic experiments. To determine whether carrier-mediated transport processes were contributing to the uptake of clonidine, kinetic experiments were performed. When the initial rates of uptake were plotted as a function of medium concentration, it became evident that overall uptake (fig. 2A, top curve) consisted of two components: (1) a linear term, which could be attributed to passive diffusion, and (2) a term that indicated Michaelis-Menten-type saturation of uptake (fig. 2B). To obtain estimates of kinetic parameters, a nonlinear regression computer program was used. The estimates for the Michaelis constants revealed a K_M value of 1.34 mM and a V_max value of 0.099 nmol/min/cm².

View larger version (K): [in this window] [in a new window]   Fig. 2.   A, Concentration dependence of clonidine uptake at room temperature () and 4°C (). B, Carrier-mediated component of uptake of clonidine at room temperature. Difference between overall uptake (A, ) and a calculated linear term representing passive diffusion is shown. Symbols are mean ± S.E.M. of 8 data points from two independent experiments. Straight lines represent calculated values of a nonlinear regression.

Mechanism of uptake. The nature of the clonidine transport mechanism was further investigated through transendothelial transport experiments (table 1). Again, translocation of clonidine was low. Earlier experiments revealed a P_app ratio of propranolol to sucrose of 3.2 (data not shown). There was no difference in transport of clonidine from apical to basolateral or in the reverse direction, suggesting that a putative carrier system is not polarized. In another set of experiments, the concentration of glucose in the transport buffer was elevated. As a result, paracellular leakage of sucrose increased by a factor of ~2. Under these conditions, P_app values of clonidine and sucrose were not significantly different (table 1). We concluded from this experiment that overall transendothelial transport of clonidine consists of a combination of passive diffusion and an additional transport mechanism that cannot be attributed to paracellular leakage.

	Discussion

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It was the aim of the present study to determine the mechanisms of transport of clonidine through the blood-brain barrier. Three lines of evidence suggest that transport of clonidine at the blood-brain barrier occurs via a carrier-mediated transport mechanism. First, uptake of clonidine by brain capillary endothelial cells showed self-inhibition (saturation) and could be described by a Michaelis-Menten-type kinetics (K_M = 1.34 mM; V_max = .099 nmol/min/cm²). Second, at a low temperature, uptake was considerably reduced and could be attributed to passive diffusion only. Third, transendothelial transport experiments suggest a contribution of active transport toward translocation of clonidine. In this case, transport experiments were performed under physiological glucose concentrations (4 mM) or in presence of 20 mM glucose. High concentrations of glucose have been reported to increase the permeability of the epithelial paracellular pathway through reversible dilatation of the paracellular space within tight junctions (Atisook and Madara, 1991; Fricker and Drewe, 1995; Madara et al., 1993). Under these conditions, P_app values of clonidine and the extracellular marker sucrose were nearly identical. This contrasts with the situation with 4 mM glucose; translocation of clonidine could not be attributed solely to paracellular leakage.

Further characterization of the putative carrier mechanism by transendothelial transport experiments revealed no difference between apical-to-basolateral transport or basolateral-to-apical transport. We therefore concluded that the carrier system responsible for transport of clonidine is localized on the apical as well as the basolateral membrane domain of cultured brain capillary endothelial cells. Uptake of clonidine into brain capillary endothelial cells was not a direct energy-requiring process and was reduced in the presence of the K⁺ ionophore valinomycin or at low extracellular pH. This marked pH sensitivity could have several causes. First, transport of clonidine could be reduced on protonation of the drug: pKa_a values are 8.10 for clonidine hydrochloride and 5.96 for clonidine base (clonidine data sheet by Boehringer Ingelheim). Second, activity of the putative carrier system could be influenced by extracellular pH, and, third, translocation of clonidine could be coupled to translocation of H⁺ from the inside of the cell to its outside. A carrier system with this characteristic and an affinity for clonidine has been described in human placenta (Ganapathy et al., 1986) and opossum kidney cells (Ramamoorthy et al., 1991). The carrier has been identified as an Na⁺/H⁺ antiporter responsible for regulation of intracellular pH in vertebrate cells (Frelin et al., 1988). Clonidine has been found to competitively inhibit Na⁺ translocation (Ganapathy et al., 1986). To test the hypothesis that clonidine might be a substrate for the Na⁺/H⁺ antiporter in brain capillary endothelial cells, uptake of clonidine was determined in the absence of extracellular Na⁺ and the presence of the specific inhibitor cimetidine (Ganapathy et al., 1986). However, in both cases, uptake of clonidine was not influenced, thus making unlikely a possible contribution of the Na⁺/H⁺ exchanger toward clonidine transport in brain capillary endothelial cells.

Transport of clonidine was compared with transport of propranolol, a reference substance that is considered to cross the blood-brain barrier via a passive diffusion process (Dehouck et al., 1992). Surprisingly, overall uptake and the P_app of clonidine were lower than expected in view of its high hydrophobicity and the involvement of a carrier system. One explanation for the lower-than-expected rate of transendothelial transport could be accumulation and resultant "trapping" of very hydrophobic substances in the hydrophobic interior of the lipid bilayer of cells (Seelig et al., 1994). This observation is corroborated by clinical data. High-dose intravenous clonidine is needed to achieve central effects; in this case, >20 mg/day of the drug is used (Tryba et al., 1993), whereas ~0.5 mg is needed for neuraxial analgesia (Goudas, 1995). In addition, there is experimental evidence that the bioavailability of clonidine in cerebrospinal fluid after intravenous injection may be almost 3 orders of magnitude lower than that after epidural administration (Castro and Eisenach, 1989). There also is a significant difference between epidural and intravenous clonidine in reducing total electroencephalographic power during general anesthesia (De Kock et al., 1992). Another explanation for the poor transport of clonidine would be the presence of a specific carrier system extruding clonidine from endothelial cells. P-glycoprotein, which confers the multidrug resistance phenotype to tumor cells, is such a transport system (Endicott and Ling, 1989; Roninson, 1992). P-glycoprotein has a broad substrate specificity and was localized at the luminal side of capillary endothelial cells in both gray matter of the brain and primary cultured bovine brain capillary endothelial cells (Tsuji et al., 1992) and the cell culture model used in the present study (Huwyler et al., 1996). However, we could exclude the possibility of an involvement of P-glycoprotein by demonstrating that vinblastine and verapamil, two well-established inhibitors of P-glycoprotein, had no effect on uptake of clonidine (data not shown).

In summary, a carrier system for clonidine has been identified using an in vitro cell culture system consisting of cultured porcine brain capillary endothelial cells. Transport was saturable and sensitive to temperature, pH and extracellular K⁺. Overall transport of clonidine was lower than expected despite this involvement of a carrier. Our data confirm clinical data indicating that clonidine is able to cross the blood-brain barrier, although relatively high doses of intravenous clonidine are needed to achieve central effects.