Inhibition of Penicillin Transport from the Cerebrospinal Fluid after Intracisternal Inoculation of Bacteria

The effect of intracisternal inoculation of bacteria on the choroid plexus system, which transports penicillin from cerebrospinal fluid (CSF) to blood, was studied in vitro and in vivo. Meningeal and choroid plexus inflammations as well as CSF pleocytosis were induced in rabbits with intracisternal inoculations of Hemophilus influenzae or Staphylococcus aureus. At various times after bacterial inoculation, the choroid plexuses of the inoculated rabbits were removed and incubated in artificial CSF containing [(14)C]penicillin. The ability of the choroid plexuses to accumulate pencillin in vitro was measured and was found to be depressed as compared with controls. This depression of choroid plexus uptake reversed with resolution of the inflammatory process. In vivo on the day after intracisternal inoculation of Hemophilus influenzae, a decrease in the disappearance of penicillin relative to inulin in the inoculated rabbits (as compared to the controls) was observed when [(14)C]penicillin and [(3)H]inulin were injected intraventricularly and cisternal CSF was sampled 2 h later. This decrease could not be explained by penicillin binding to the CSF exudate. However, the choroid plexus transport system for penicillin was only partially depressed in those inoculated rabbits with bacterially induced inflammation, since in vitro the choroid plexuses could still accumulate penicillin and in vivo CSF penicillin levels could be further increased with probenecid pretreatment. These results suggest that CSF penicillin levels are increased in this model due to three factors: a depression of active efflux of penicillin from the CSF, an increase in permeability to penicillin of inflamed meninges, and, less significantly, by CSF binding of penicillin.     

Riboflavin transport in the central nervous system. Characterization and effects of drugs.

The relationship of riboflavin transport to the transport of other substances including drugs in rabbit choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, and brain cells were studied in vivo and in vitro. In vitro, the ability of rabbit choroid plexus to transport riboflavin from the medium (cerebrospinal fluid surface) through the choroid plexus epithelial cells into the extracellular and vascular spaces of the choroid plexus was documented using fluorescence microscopy. These studies provided further evidence that riboflavin is transported from cerebrospinal fluid to blood via the choroid plexus. The transport of [14C]riboflavin by the isolated choroid plexus was inhibited by thiol agents, ouabain, theophylline, various flavins (lumiflavin and lumichrome > sugar containing flavins), and cyclic organic acids including penicillin and fluorescein. Riboflavin inhibited [14C]penicillin transport competitively and the inhibition constant (K1) for riboflavin equaled the concentration of riboflavin at which the saturable transport system for riboflavin is 50% saturated (KT). These and other data suggest that riboflavin, penicillin, and possibly fluorescein are transported by the same transport system in choroid plexus. In vivo, the intra-ventricular injection or riboflavin and [14C]penicillin inhibited [14C]penicillin transport from cerebrospinal fluid. In vitro, various flavins (riboflavin > other sugar-containing flavins > lumiflavin > lumichrome) inhibited [14C]riboflavin accumulation by brain slices. These studies support the notions that: (a) riboflavin accumulation by choroid plexus (active transport) is quite different from that in brain cells (facilitated diffusion and intracellular trapping), and (b) therapeutically important cyclic organic acids (e.g., penicillin) are transported fom cerebrospinal fluid by the riboflavin transport system in choroid plexus.     

Transport of diphenhydramine in the central nervous system

The transport and metabolism of diphenhydramine was studied in vitro in the isolated rabbit choroid plexus and in vivo in New Zealand white rabbits and Sprague-Dawley rats. In vitro, [14C] diphenhydramine was accumulated by a saturable, energy-requiring system in choroid plexus. In vivo, 20 min after intraventricular injection into rabbits, [14C]diphenhydramine was cleared from cerebrospinal fluid much more rapidly than [3H]sucrose, a molecule transported in the central nervous system by simple diffusion. In vivo, employing the in situ rat brain perfusion technique, [14C]diphenhydramine was cleared from the cerebral perfusion fluid as rapidly as [14C]diazepam. However, the clearance of [14C]diphenhydramine, but not [14C]diazepam, was inhibited by the addition of 10 mM unlabeled diphenhydramine to the perfusate. These in vivo and in vitro results show that diphenhydramine, unlike diazepam, is transported between blood, brain and cerebrospinal fluid, in part, by saturable, carrier-mediated transport processes at both the blood-brain and blood-cerebrospinal fluid barriers.     

Ceftriaxone pharmacokinetics in the central nervous system

The transport and metabolism of ceftriaxone was studied in vitro in the isolated choroid plexus and in vivo in New Zealand White rabbits. In vitro, [14C]ceftriaxone was accumulated by a saturable, probenecid-sensitive system in choroid plexus, although much less readily than [14C]penicillin G. Ceftriaxone was also a much less potent inhibitor of [14C]penicillin G accumulation by the isolated choroid plexus than penicillin G itself (IC50 = 1.6 vs. 0.07 mM, respectively). In vivo, 2 hr after intraventricular injection, [14C]ceftriaxone was not metabolized or cleared from the cerebrospinal fluid more rapidly than [3H]mannitol, a molecule transported in the central nervous system by simple diffusion. These in vitro and in vivo results show that ceftriaxone, unlike penicillin G, has minimal affinity for the choroid plexus active transport system that transfers most penicillins and cephalosporins from cerebrospinal fluid to blood.     

The effect of uremia on penicillin flux between blood and cerebrospinal fluid.

The effect of uremia on the choroid plexus system responsible for transport of penicillin from cerebrospinal fluid (CSF) to blood was studied in vitro and in vivo. Uremia was induced in rabbits by injecting toxic doses of cephaloridine or by obstructing urine flow. Three days after the induction of uremia, isolated choroid plexuses from normal rabbits were unable to concentrate penicillin 14C normally when incubated in either CSF or ultrafiltrates of plasma from uremic rabbits. In vivo, a decrease in the disappearance of penicillin 14C from CSF was observed in uremic rabbits. However, the choroid plexus transport system for penicillin was only partially and reversibly depressed in uremia. The increased CSF levels of penicillin uremia are due to: decreased excretion of penicillin by the kidney, depression of active efflux of penicillin from the CSF, and decreased plasma binding of penicillin.     

The transport of gentamicin in the choroid plexus and cerebrospinal fluid.

Gentamicin often attains inadequate therapeutic levels in cerebrospinal fluid (CSF). In the present study, the possibility that gentamicin might be transported from CSF to blood by a mechanism in the choroid plexus was investigated. Rabbit choroid plexuses were incubated 15 to 120 minutes in artificial CSF containing 14C-gentamicin and 3H-inulin. The choroid plexuses accumulated gentamicin linearly up to 120 minutes where a tissue/medium ratio of 5.4 was attained. Uptake of gentamicin was inhibited by: 1) increasing the gentamicin concentration or adding kanamycin; 2) omitting oxygen and glucose; 3) adding dinitrophenol to the medium; 4) incubating at 4 degrees C; OR 5) incubating in rabbit CSF. However, uptake was not inhibited by probenecid or ammonium ion. Kinetic analysis revealed that gentamicin was transported into choroid plexus by a process that satisfied Michaelis-Menten kinetics. A Lineweaver-Burk transformation of the transport values for the saturable component yielded a Kt of 51.8 mug/ml and a Vmax = 1.8 mug/ml/min. In vivo, gentamicin clearance from CSF, although saturable, was only 1.4 times greater than inulin because of the relatively low Kt and Vmax of the choroid plexus transport system for gentamicin and the presence of inhibitors of gentamicin transport in rabbit CSF. We conclude that the saturable transport of gentamicin from CSF, although quantitatively not large, is a factor in determining gentamicin levels in CSF.     

Comparative pharmacology of the nitrobenzylthioguanosine-sensitive and -resistant nucleoside transport mechanisms of Ehrlich ascites tumor cells

A variety of nucleoside transport inhibitors and substrates were compared for their capacities to inhibit the zero-trans influx of [3H]uridine in Ehrlich ascites tumor cells. ATP-depleted cells accumulated [3H]uridine primarily by facilitated diffusion (Vmax = 16 pmol/sec/microliter cell water) via both nitrobenzylthioguanosine (NBTGR)-sensitive (IC50 = 0.53 nM, 100 microM [3H]uridine) and NBTGR-resistant (IC50 = 71 microM, 100 microM [3H]uridine) mechanisms with uridine Km estimates of 99 and 284 microM, respectively. Dilazep also distinguished between the transporter subtypes with IC50 values of 1.4 nM and 1.8 microM, respectively, for inhibiting 100 microM [3H]uridine influx. Incubation of cells with 50 nM NBTGR allowed the selective study of inhibitor effects on NBTGR-resistant [3H]uridine influx. Dipyridamole, cyclopentyladenosine, 2-phenylaminoadenosine, etoposide, teniposide, diazepam, chlordiazepoxide, triazolam and the lidoflazine derivative 2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis-(4- fluorophenyl)pentyl]-1-piperazineacetamide (R75231), were significantly less potent as inhibitors of NBTGR-resistant influx, when compared with their capacities to inhibit the total mediated influx of [3H]uridine. In contrast, 2-fluoroadenosine, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine and soluflazine were relatively more effective as inhibitors of the NBTGR-resistant component. Mioflazine, a compound related to both soluflazine and R75231, did not distinguish between transporter subtypes. The NBTGR-resistant transporter also had a distinctive substrate specificity; guanosine, 2'-deoxyguanosine, cytidine and 2'-deoxycytidine were significantly less effective as inhibitors of NBTGR-resistant [3H]uridine influx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active transport of fentanyl by the blood-brain barrier

Previous studies have shown that uptake of the lipophilic opioid, fentanyl, by pulmonary endothelial cells occurs by both passive diffusion and carrier-mediated processes. To evaluate if the latter mechanism also exists in brain endothelium, transport of [3H]fentanyl was examined in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers. Uptake of fentanyl appears to occur via a carrier-mediated process as uptake of [3H]fentanyl by BBMECs was significantly inhibited in a dose-dependent manner by unlabeled fentanyl. Fentanyl uptake was also significantly inhibited by either 4 degrees C or sodium azide/2-deoxyglucose, suggesting that carrier-mediated uptake of fentanyl was an active process. Fentanyl was also tested to determine whether it might be a substrate of the endogenous blood-brain barrier efflux transport system, P-glycoprotein (P-gp). Release of [3H]fentanyl or rhodamine 123, a known substrate of P-gp, previously loaded in the BBMECs was studied in the presence or absence of either fentanyl or verapamil, a known competitive inhibitor of P-gp. Both fentanyl (10 microM) and verapamil (100 microM) decreased release of rhodamine 123 from BBMECs, indicating that fentanyl is a substrate of P-gp in the BBMECs. This was further supported by the observation that uptake of [3H]fentanyl was significantly increased in Mg2+-free medium, a condition known to reduce P-gp activity. However, release of [3H]fentanyl was significantly increased when incubated with either unlabeled fentanyl or verapamil. These results suggest that the active P-gp-mediated extrusion of fentanyl in these cells is overshadowed by an active inward transport process, mediated by an as yet unidentified transporter. In addition, verapamil was shown to be a substrate of both P-gp and the fentanyl uptake transporter.     

Anion exchanger mediates benzylpenicillin transport in rat choroid plexus

Transport characteristics of benzylpenicillin in the central nervous system was examined using ATP-depleted rat choroid plexus. In the presence of an outwardly directed Cl- gradient, accumulation of benzylpenicillin was stimulated markedly compared with the accumulation in the absence of Cl- gradient. Under Cl- gradient conditions, benzylpenicillin was accumulated transiently at a concentration approximately 1.7-fold higher than at equilibrium ("overshoot") implying the uphill transport. The Cl- gradient-stimulated accumulation of benzylpenicillin was not solely due to the changes in the membrane potential or in the intracellular pH. Accumulation of benzylpenicillin in choroid plexuses preloaded with HCO3-, SCN- or benzylpenicillin also was stimulated compared with the accumulation in unpreloaded choroid plexuses. The Cl- gradient-stimulated benzylpenicillin accumulation was saturable (Km = 13.6 microM and Vmax = 3.76 nmol/ml of tissue per min), and was reduced by sulfhydryl reagents (p-hydroxymercuribenzoic acid, p-chloromercuribenzoic acid, p-chloromercuribenzenesulfonic acid and N-ethylmaleimide). Probenecid and anionic exchange inhibitors (furosemide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) inhibited the Cl- gradient-stimulated benzylpenicillin accumulation in a dose-dependent fashion. An inwardly directed Na+ gradient did not stimulate the accumulation of benzylpenicillin. These findings suggest that the principal mechanism for the uphill transport of benzylpenicillin in the rat choroid plexus is via an anionic exchanger.     

Distribution of suramin, an antitrypanosomal drug, across the blood-brain and blood-cerebrospinal fluid interfaces in wild-type and P-glycoprotein transporter-deficient mice

Although 60 million people are exposed to human African trypanosomiasis, drug companies have not been interested in developing new drugs due to the lack of financial reward. No new drugs will be available for several years. A clearer understanding of the distribution of existing drugs into the brains of sleeping sickness patients is needed if we are to use the treatments that are available more safely and effectively. This proposal addresses this issue by using established animal models. Using in situ brain perfusion and isolated incubated choroid plexus techniques, we investigated the distribution of [(3)H]suramin into the central nervous systems (CNSs) of male BALB/c, FVB (wild-type), and P-glycoprotein-deficient (Mdr1a/Mdr1b-targeted mutation) mice. There was no difference in the [(3)H]suramin distributions between the three strains of mice. [(3)H]suramin had a distribution similar to that of the vascular marker, [(14)C]sucrose, into the regions of the brain parenchyma that have a blood-brain barrier. However, the association of [(3)H]suramin with the circumventricular organ samples, including the choroid plexus, was higher than that of [(14)C]sucrose. The association of [(3)H]suramin with the choroid plexus was also sensitive to phenylarsine oxide, an inhibitor of endocytosis. The distribution of [(3)H]suramin to the brain was not affected by the presence of other antitrypanosomal drugs or the P-glycoprotein efflux transporter. Overall, the results confirm that [(3)H]suramin would be unlikely to treat the second or CNS stage of sleeping sickness.     

Differential effects of acetazolamide, benzolamide and systemic acidosis on hydrogen and bicarbonate gradients across the apical and basolateral membranes of the choroid plexus

The effect after 1 hr of sulfonamide agents and systemic acidosis on pH (dimethyloxazolidinedione method) and [HCO3-] (calculated from pH and estimated cell pCO2) of choroid plexus epithelium was analyzed in adult Sprague-Dawley rats anesthetized with ether. Acetazolamide (20 mg/kg i.p.) caused a striking increase in choroid cell pH (7.0-7.45) and a substantial elevation in [HCO3-]i, and an alkalinization of cerebrospinal fluid (CSF); effects of benzolamide (3 mg/kg i.v.) were less marked. Acetazolamide-induced augmentation of steady-state pH was found in choroid plexus (0.45 pH unit) but not in submaxillary salivary gland, skeletal muscle, erythrocytes and cerebral cortex. Respiratory acidosis (blood pH 6.94) caused choroid cell pH to decrease less extensively than in the other tissues analyzed; metabolic acidosis (blood pH 7.23) did not significantly alter choroid plexus pH. Both sulfonamide agents and systemic acidoses significantly decreased the pH gradient between arterial blood and cisternal CSF. The sulfonamide-induced increase in choroid cell pH is attributed to suppression of buffering of OH- by CO2, leading to elevated [OH]i; and to a build-up in [HCO3-]i. Acetazolamide caused a reversal of the H+ and HCO3- gradients across the basolateral (plasma-facing) membrane of the choroid plexus. It is postulated that carbonic anhydrase inhibitors decrease CSF formation primarily by reducing Na+-H+ exchange at the basolateral membrane of the choroidal epithelium, and secondarily by slowing down HCO3- and Cl- exit across the CSF-facing membrane.     

Transport of imipenem, a novel carbapenem antibiotic, in the rat central nervous system

The transport of imipenem, a novel carbapenem antibiotic, in the rat central nervous system (CNS) was studied using in vivo, in situ and in vitro experimental techniques. After i.v. bolus administration, the imipenem concentration in the cerebrospinal fluid (CSF) rose to a peak within 30 min and declined with time. The CSF/serum unbound concentration ratio of imipenem was 0.22 at 2 hr after i.v. administration, substantially higher than that reported for benzylpenicillin. By using an in situ brain perfusion technique, we found that imipenem was transported through the blood-brain barrier principally via passive diffusion with a permeability-surface area product comparable to that of mannitol. In vitro, imipenem was accumulated by the isolated choroid plexus via an active organic anion transport system, although much less rapidly than benzylpenicillin. In vivo, after i.c.v. administration, imipenem was cleared from the CNS in a manner comparable to that of mannitol with only a small probenecid-sensitive process. Imipenem thus has minimal affinity for the organic anion transport system in the choroid plexus, resulting in the slow elimination of this drug from the CNS. These results suggest that the difference between imipenem and benzylpenicillin in the ratio of CSF to unbound serum drug concentration is determined principally by the efflux process in the choroid plexus rather than the influx process through the blood-brain barrier.     

3H]tryptamine autoradiography in rat brain and choroid plexus reveals two distinct sites

In vitro autoradiographic techniques were used to examine the distribution of [3H]tryptamine binding sites in rat brain. The gross distribution and pharmacological characteristics of binding to brain sections resembled those seen in homogenate studies. Binding sites were found throughout the brain, with a preponderance of sites in the forebrain and limbic structures; highest levels were seen in the choroid plexus and the interpeduncular nucleus. Other regions exhibiting high levels of [3H]tryptamine binding include the cortex (especially lamina I), caudate putamen, hippocampus, anterior olfactory nucleus, olfactory tubercle, nucleus accumbens, amygdala, superior colliculus (superficial gray layer), locus ceruleus, the nucleus of the solitary tract and the pineal body. Although there were similarities in this distribution to that for binding sites of [3H]5-hydroxytryptamine ([3H]serotonin), the overall patterns were distinct. The binding site for [3H] tryptamine in the choroid plexus (termed T-2) was pharmacologically distinct from that in the rest of the brain (termed T-1); several compounds, including kynuramine, were potent inhibitors of [3H]tryptamine binding at the brain site, but not at the choroid plexus site. [3H]Serotonin also labels a site in the rat choroid plexus; this site was different from both [3H]tryptamine sites. Knowledge of the distribution of tryptamine binding sites in the brain will aid in efforts to ascertain the function of these sites.     

A kinetic study of the ouabain-induced efflux of norepinephrine from the dog saphenous vein

Dog saphenous vein strips were incubated with [3H]norepinephrine ([3H]NE), 1.4 microM, after inhibition of the NE-metabolizing enzymes and extraneuronal uptake, and superfused for up to 290 min. From the 70th min onwards the strips were exposed to 10 microM ouabain, some of them being subject to electrical stimulation from the 140th min onwards. Other strips were exposed to either 1, 10 or 100 microM ouabain from the 70th min onwards. The spontaneous efflux of [3H]NE had a long half-time (156 min), and over 90% of the [3H]NE accumulated did not participate in efflux ("bound fraction"). Ouabain, 10 microM, induced a pronounced increase of the rate of efflux of [3H]NE, which was delayed in its onset and reached a maximum at t = 135 min of superfusion. Increasing the concentration of ouabain decreased both the delay to the beginning of the overflow and the time to maximum efflux and increased the maximum rate of efflux. In Ca(++)-free medium (during the superfusion period), the maximum rate of efflux was lower than in Ca(++)-containing medium, but was attained earlier. The bound fraction amounted to 22% when the efflux was induced by 10 microM ouabain in Ca(++)-containing medium, a value unnaffected by electrical stimulation but reduced markedly by omitting calcium. The results support the view that the efflux of [3H]NE induced by ouabain is delayed and that it is both carrier-mediated and due to exocytosis.     

Erythromycin and azithromycin transport into Haemophilus influenzae ATCC 19418 under conditions of depressed proton motive force (delta mu H).

The effect of collapsing the electrochemical proton gradient (delta mu H) on [3H]erythromycin and [14C]azithromycin transport in Haemophilus influenzae ATCC 19418 was studied. The proton gradient and membrane potential were determined from the distribution of [2-14C]dimethadione and rubidium-86, respectively. delta mu H was reduced from 124 to 3 mV in EDTA-valinomycin-treated cells at 22 degrees C with 150 mM KCl and 0.1 mM carbonyl cyanide m-chlorophenylhydrazone. During the collapse of delta mu H, macrolide uptake increased. Erythromycin efflux studies strongly suggested that this increase was not due to an energy-dependent efflux pump but was likely due to increased outer membrane permeability. These data indicated that macrolide entry was not a delta mu H-driven active transport process but rather a passive diffusion process.     

Effects of kelatorphan and other peptidase inhibitors on the in vitro and in vivo release of methionine-enkephalin-like material from the rat spinal cord

The effects of the novel mixed peptidase inhibitor, kelatorphan [N-(R)-3-(N-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)-L-alanine], were compared to those of a combination of the potent "enkephalinase" inhibitor thiorphan and the nonselective aminopeptidase inhibitor bestatin, on the catabolism of [3H]Met-enkephalin and on the release of endogenous Met-enkephalin by the rat spinal cord in vitro and in vivo. At 20 microM, kelatorphan almost prevented completely the degradation of exogenous [3H] Met-enkephalin by slices of the dorsal zone of the lumbar enlargement. Similarly, the addition of 20 microM kelatorphan to a [3H] Met-enkephalin-containing artificial cerebrospinal fluid superfusing the whole spinal cord of halothane-anesthetized rats efficiently protected the exogenous peptide from enzymatic degradation. In contrast, in the same in vitro and in vivo models, thiorphan (1 microM) or bestatin (20 microM) alone was inactive, and only their combination induced a significant protection of the exogenous peptide. In vitro and in vivo, kelatorphan (20 microM) increased markedly the spontaneous outflow of endogenous Met-enkephalin-like material as well as the peptide overflow due to K+-induced depolarization (in vitro and in vivo) or noxious stimulation (in vivo). Under similar conditions, thiorphan (1 microM) plus bestatin (20 microM) also enhanced the efflux of Met-enkephalin-like material, but generally to a lower extent than kelatorphan. Compared to thiorphan plus bestatin, kelatorphan exerts additional inhibitory effects on dipeptidylaminopeptidase activity and the present results could indicate that this enzyme also may be involved in the inactivation of extracellular Met-enkephalin at the spinal level in rats.     

Effects of amiloride and 5-(N,N-hexamethylene)amiloride on dopaminergic and cholinergic neurotransmission

In order to establish the role of the Na+/H+ exchange transport on neurotransmission, we investigated the effects of amiloride and of 5-(N,N-hexamethylene)amiloride (HMA) on dopamine (DA) and acetylcholine (ACh) release and on receptor-mediated modulation of DA and ACh release. Superfused rabbit striatal slices prelabeled with [3H]DA and [14C]choline were stimulated electrically in the presence and absence of several concentrations of these agents. Amiloride (3-10 microM) and HMA (0.3-10 microM) reduced the basal efflux and the stimulation evoked overflow of total 3H and of [3H]-3,4-dihydroxyphenylacetic acid and inhibited monoamine oxidase activity. The inhibition of stimulation evoked overflow of total 3H was blocked by pretreatment with nomifensine but not by sulpiride. Amiloride had no effect on the basal efflux and the stimulation evoked overflow of ACh or it did modify apomorphine-induced inhibition of DA and ACh release. However, at 3 to 10 microM, HMA enhanced the basal efflux of 3H; this effect was not prevented either by uptake inhibition with nomifensine or by low extracellular calcium. These results suggest that amiloride-sensitive Na+ transport and the amiloride and HMA-sensitive Na+/H+ antiporter play no role on the secretion of DA and ACh, or on the mechanisms by which activation of pre- and postsynaptic DA receptors lead to inhibition of neurotransmitter release. Amiloride- and HMA-induced monoamine oxidase inhibition accounts for the effects of amiloride and HMA on DA efflux and overflow. The guanidine moiety present in the amiloride and HMA molecules is most likely responsible for these effects.