Muscarinic modulation of hypoxia-induced release of catecholamines from the cat carotid body.

Chemotransduction of arterial hypoxemia by the cat carotid body is generally thought to begin with a hypoxia-induced depolarization of the glomus cells (GCs) of the carotid body (CB). This depolarization activates voltage-gated calcium channels with the subsequent entry of calcium, movement of transmitter-containing vesicles to the synaptic-like juncture between the GC and apposed sensory afferent neuron. The vesicles exocytotically release their transmitters which then proceed to the receptors on both the postsynaptic neuron and on the GCs themselves (autoreceptors). Action potentials and their modulation in the sensory fibers are the result, along with the modulation of further neurotransmitter release from the GCs. The purpose of the present study was to: (1) determine the parameters of an incubated cat CB preparation capable of releasing measurable amounts of catecholamines (CAs) in response to hypoxia; (2) determine the impact of muscarinic activities on CA release during the hypoxic challenge; (3) determine if the muscarinic activity preferentially modified the release of one CA more than another; (4) determine if there were any differences in the pattern of hypoxia-induced release of dopamine (DA) vs. norepinephrine (NE). CBs were harvested from deeply anesthetized cats. Cleaned of fat and connective tissue, they were incubated in Krebs Ringer bicarbonate solution at 37 degrees C, and bubbled with a hyperoxic mixture of gases (95% O(2)-5% CO(2)) for 30 min. The first series of experiments to address the CB's hypoxia-induced release of CAs explored the effects of incubating CBs for 2 h with hyperoxia vs. normoxia (21% O(2)-6% CO(2)) followed by a 30 min hypoxic challenge, with or without L-dihydroxyphenylalanine (L-DOPA). In the second series of experiments the CBs, after the first 30 min of hyperoxia, were next challenged with hypoxia (4% O(2)-5% CO(2)) for intervals of 3-20 min with intervening recovery periods of hyperoxia to determine the effect of the duration of the hypoxic exposure on CA release. In the third series of experiments the CBs, after the first 30 min of hyperoxia, were challenged with hypoxia for intervals of 10-40 min in the presence or absence of an M1 or M2 muscarinic receptor antagonist. CAs released into the incubation medium were analyzed by means of high performance liquid chromatography-electrochemical detection using standard procedures. Incubated cat CBs challenged for 2 h with hyperoxia followed by 30 min of hypoxia, released much more measurable amounts of CAs in the presence of 40 microM L-DOPA than without it. Moving from hyperoxia to hypoxia produced a better yield than moving from normoxia to hypoxia, and at least 10-20 min exposures were needed for measurable amounts of CAs. The M1 muscarinic receptor antagonist, pirenzepine, reduced the hypoxia-induced release of CAs during each exposure. Further, the reduction appeared to be dose-related. The M2 muscarinic receptor antagonist, methoctramine, enhanced the hypoxia-induced release of CAs during each exposure. These data support a role for acetylcholine (ACh) in the hypoxia-induced release of CAs, and suggest a significant, if modest, muscarinic dimension to it. And although hypoxia induced a greater release of DA than of NE, the muscarinic modulation of the release (both decreasing it and increasing it) may have had a greater impact on NE release than on DA release. Finally, the patterns of hypoxia-induced release of DA and NE from incubated cat carotid bodies are significantly different.

The impact of adenosine on the release of acetylcholine, dopamine, and norepinephrine from the cat carotid body.

Exogenously administered adenosine provokes an increase in respiration in both animal models and in man. Administered near the carotid body adenosine increases neural output from the carotid body in rats and cats. Hypoxia has the same effect. Hypoxia also provokes a release of acetylcholine (ACh), dopamine (DA), and norepinephrine (NE) from the carotid body. The present study aimed to determine the effect of exogenous adenosine on the release of ACh, DA, and NE from the carotid bodies of cats. After a recovery period (from surgery) carotid bodies were first incubated for 10 (DA, NE) or 15 (ACh) min in Eppendorf tubes containing 85 microL of a physiological salt solution equilibrated with 40% O2/5% CO2 at 37 degrees C (hyperoxia). At the end of the incubation period the medium was drawn off, and measured for ACh, DA, and NE using HPLC-ECD methods. Next 85 microL of the medium and the tubes were equilibrated with a hypoxic gas mixture (4% O2/5% CO2) and the carotid bodies were incubated for 10 (DA, NE) or 15 (ACh) min, at the end of which the medium was drawn off and measured for ACh, DA, and NE. In the ACh studies there followed a post-hypoxic hyperoxic exposure (40% O2/5% CO2). ACh tubes were then made 100 microM with respect to adenosine, and the hyperoxic, hypoxic, and post-hypoxic hyperoxic challenges were repeated. One of the two DA, NE tubes had the 100 microM adenosine from the start. Adenosine significantly increased the release of ACh, but significantly decreased the hypoxia-induced release of DA. Potential mechanisms for these changes are reviewed.

Pharmacodynamic and therapeutic investigation of focused ultrasound-induced blood-brain barrier opening for enhanced temozolomide delivery in glioma treatment.

Focused ultrasound (FUS) exposure with the presence of microbubbles has been shown to transiently open the blood-brain barrier (BBB), and thus has potential to enhance the delivery of various kinds of therapeutic agents into brain tumors. The purpose of this study was to assess the preclinical therapeutic efficacy of FUS-BBB opening for enhanced temozolomide (TMZ) delivery in glioma treatment. FUS exposure with microbubbles was delivered to open the BBB of nude mice that were either normal or implanted with U87 human glioma cells. Different TMZ dose regimens were tested, ranging from 2.5 to 25 mg/kg. Plasma and brain samples were obtained at different time-points ranging from 0.5 to 4 hours, and the TMZ concentration within samples was quantitated via a developed LC-MS/MS procedure. Tumor progression was followed with T2-MRI, and animal survival and brain tissue histology were conducted. Results demonstrated that FUS-BBB opening caused the local TMZ accumulation in the brain to increase from 6.98 to 19 ng/mg. TMZ degradation time in the tumor core was found to increase from 1.02 to 1.56 hours. Improved tumor progression and animal survival were found at different TMZ doses (up to 15% and 30%, respectively). In conclusion, this study provides preclinical evidence that FUS-BBB opening increases the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting the potential for clinical application to improve current brain tumor treatment.

Focused ultrasound-induced blood-brain barrier opening to enhance temozolomide delivery for glioblastoma treatment: a preclinical study.

The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.

Inhibition of sphingosine 1-phosphate lyase for the treatment of rheumatoid arthritis: discovery of (E)-1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethanone oxime (LX2931) and (1R,2S,3R)-1-(2-(isoxazol-3-yl)-1H-imidazol-4-yl)butane-1,2,3,4-tetraol (LX2932).

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

Inhibition of sphingosine-1-phosphate lyase for the treatment of autoimmune disorders.

During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.