5-hydroxytryptamine3 (5-HT3) receptors mediate spinal 5-HT antinociception: an antisense approach.

To examine the role of the 5-hydroxytryptamine(1B) (5-HT1B) and 5-HT3 receptor subtypes in the analgesia produced by 5-HT (serotonin) agonists, we assessed the effect of antisense oligodeoxynucleotides (AODNs) designed to "knock down" the number of these receptor subtypes on analgesia produced by intrathecal (i.t.) 5-HT, the 5-HT1B receptor agonist, 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline maleate (CGS-12066A), and the 5-HT3 receptor agonist, 2-methyl-5-HT. Groups of mice (n = 17-20) were injected i.t. on days 1, 3, and 5 with one of the AODNs, a mismatch oligo, or saline. On day 6, all mice were injected i.t. with 70.5 nmol of 5-HT, 44.4 nmol of CGS-12066A, or 49 nmol of 2-methyl-5-HT by lumbar puncture. Following testing, spinal cords were rapidly removed and prepared for receptor binding assays. Treatment with AODN for 5-HT1B receptors produced a 70% reduction in ligand binding to this receptor subtype. After treatment with AODN for 5-HT3 receptors, ligand binding to this receptor subtype was undetectable. In mice tested with i.t. 5-HT, tail-flick analgesia was attenuated only in mice treated with the 5-HT3 receptor AODN. Mice treated with the AODN designed to knock down 5-HT(1B) receptors or with its mismatch oligo were not significantly different from controls. In mice tested with i.t. administration of CGS-12066A, none of the oligo treatments produced a significant attenuation of analgesia. In mice tested with i.t. administration of 2-methyl-5-HT, only 5-HT3 receptor AODN attenuated analgesia. Thus, 5-HT and 2-methyl-5-HT analgesia are mediated by the 5-HT3 receptor subtype. However, spinal CGS-12066A analgesia appears not to be mediated by either the 5-HT1B or the 5-HT3 receptor subtypes.

The effects of postmortem delay on mu, delta and kappa opioid receptor subtypes in rat brain and guinea pig cerebellum evaluated by radioligand receptor binding.

Studies of human and animal central nervous system receptor degradation and measurement postmortem are important as human tissue can rarely be collected under ideal experimental conditions. Correlating the change in binding of opioid receptor subtypes over time will help define the conditions under which human studies may be valid. The present study was designed to investigate the rate at which opioid receptor subtypes degrade postmortem. Brains from rats or cerebelli from guinea pigs were kept at 22 degrees C or 4 degrees C at times from zero to 24 hours to simulate two common human collection techniques; room temperature and morgue refrigeration. Tissue homogenates from these brains were analysed for mu1, mu2, delta, kappa1 and kappa3 opioid receptor binding using standard radioligand binding techniques. At room temperature mu1, mu2 and delta opioid receptor binding was reduced between 6 and 12 hours, whereas kappa1 and kappa3 binding was reduced after 24 hours. At 4 degrees C mu1, mu2, delta, kappa1 and kappa3 binding remained constant over the 24 hour period.

Effects of kappa-opioid receptor agonists on stimulated phosphoinositide hydrolysis in rat kidney.

To determine the effects of kappa-opioid receptor agonists on phosphoinositide metabolism in rat renal cortex, tissue slices labelled with [3H]inositol were stimulated with norepinephrine or carbachol alone or in combination with the kappa-opioid receptor agonists, ethylketocyclazocine, trans-3,4-dichloro-N-methyl-N-[2-(pyrrolindinyl)-cyclohexyl)- benzeneacetamide (U50,488) and nalorphine. Both norepinephrine and carbachol stimulated phosphoinositide hydrolysis (measured in a LiCl buffer) concentration- and time-dependently. The EC50 and maximal stimulation of phosphoinositide hydrolysis for norepinephrine and carbachol were approximately 3 microM and 0.15 dpm released/dpm incorporated, respectively. Concentrations up to 1 mM of ethylketocyclazocine, U50,488 or nalorphine alone did not affect phosphoinositide hydrolysis. However, ethylketocyclazocine and U50,488 decreased 10 microM norepinephrine-stimulated phosphonositide hydrolysis concentration-dependently, each with an approximate IC50 of 30 microM. In contrast, nalorphine had no effect on norepinephrine-stimulated phosphoinositide hydrolysis. In addition, concentrations of up to 1 mM ethylketocyclazocine or U50,488 did not alter carbachol-stimulated phosphoinositide hydrolysis. The inhibitory effect of U50,488 and ethylketocyclazocine on norepinephrine-stimulated phosphoinositide hydrolysis was blocked by the selective kappa-opioid receptor antagonist, nor-binaltorphimine. These results indicate that kappa 1-opioid receptor stimulation may affect phosphoinositide metabolism in rat renal cortex by modulating the subcellular effects of renal alpha 1-adrenoceptor activation.

Calcium channel involvement in potassium depolarization-induced phosphoinositide hydrolysis in rat cortical slices.

The stimulation of production of inositol phosphates in rat cortical slices by KCl depolarization and the effects of calcium channel active drugs were investigated. Elevation of K+ in the medium up to 48 mM KCl caused a linear concentration-dependent increase in [3H]inositol phosphate accumulation. The KCl stimulated response was not significantly inhibited in the presence of muscarinic or alpha 1-adrenergic antagonists. KCl stimulated the production of inositol trisphosphate at 60 min but not 10 min. Addition of peptidase inhibitors did not significantly affect KCl-stimulated PI hydrolysis. The KCl-stimulated response was still observed in the absence of extracellular calcium, although the net accumulation of inositol phosphates was greater in the presence of 0.1 or 0.5 mM calcium. KCl (48 mM) inhibited [3H]inositol uptake into phospholipids of cortical slices. The dihydropyridine calcium channel agonist BAY K 8644 stimulated PI hydrolysis in cortical slices in a concentration dependent manner in the presence of 19 mM KCl. The BAY K 8644-stimulated PI response was partially inhibited by 1 microM atropine but not by 1 microM prazosin. Calcium channel blockers nitrendipine, verapamil, flunarizine, and nifedipine slightly inhibited the PI response stimulated by 19 mM KCl in the presence or absence of BAY K 8644. The effects of the calcium channel antagonists were attenuated in the presence of 1 microM atropine. The peptide calcium channel blocker omega-conotoxin did not affect KCl-stimulated PI hydrolysis. These results suggest that endogenous muscarinic or adrenergic neurotransmitters are not involved in KCl-stimulated PI hydrolysis in cortical slices. Although extracellular calcium is necessary for optimal KCl-stimulated PI hydrolysis, it is not required for the expression of the KCl-evoked response suggesting that depolarization is the primary trigger for this stimulant.

Metabolic activation of the pesticide azinphos-methyl by perfused mouse livers.

Perfusion of mouse livers in situ with the phosphorodithioate pesticide azinphos-methyl (O,O-dimethyl S-[4-oxo-1,2,3-benzotriazin-3(4H)-ylmethyl] phosphorodithioate; Guthion) resulted in the appearance of the cholinesterase inhibitor azinphos-methyl oxon in effluent perfusate. Since mouse whole blood did not have the capacity to detoxify this toxic oxon rapidly enough to prevent its passage to extrahepatic tissues in vivo, the liver is likely a major source of azinphos-methyl oxon in the mouse following exposure to azinphos-methyl. Alterations in perfusate flow rates in situ had little effect on the hepatic disposition of azinphos-methyl. Conversely, significant increases in the free fraction of azinphos-methyl in perfusate led to marked changes in hepatic distribution and biotransformation of this pesticide. Phenobarbital pretreatment of mice induced hepatic cytochrome P-450 content, as well as microsomal activation of azinphos-methyl in vitro, yet antagonized the acute toxicity of this pesticide in vivo. Interestingly, perfused livers from phenobarbital-pretreated mice produced less azinphos-methyl oxon than perfused livers from saline-pretreated mice, thereby accounting for the antagonism of the acute toxicity of azinphos-methyl afforded by phenobarbital pretreatment. The mechanism of this phenobarbital-dependent decrease in appearance of azinphos-methyl oxon in effluent perfusate is unclear. However, it must be emphasized that the hepatic biotransformation of azinphos-methyl is complex, involving several sequential and simultaneous pathways, all of which could be affected by phenobarbital. The metabolic profile observed in effluent perfusate is the net result of all these pathways operating in the intact liver.

Factors affecting the biotransformation of the pesticide parathion by the isolated perfused mouse liver.

Single-pass perfusion of mouse livers in situ with the phosphorothioate pesticide parathion resulted in formation of paraoxon, p-nitrophenol, p-nitrophenyl sulfate, and p-nitrophenyl-beta-D-glucuronide. At a perfusate bovine serum albumin (BSA) concentration of 4% (fraction of unbound parathion = 0.04), and a flow rate of 3.2 ml/min/liver, the half-life associated with the approach to steady state of parathion was 6.2 min (SD = 0.4), whereas at steady state the extraction ratio of parathion was 0.19 (SD = 0.03). Alterations in perfusate flow rates had no discernable effects on metabolism of parathion. However, lowering the BSA perfusate concentration to 1.0% (fraction of unbound parathion = 0.12) significantly prolonged the half-life for the approach to steady state while increasing the steady state extraction ratio to 0.49 (SD = 0.08). At perfusate BSA concentrations below 1%, steady state conditions with respect to parathion could not be achieved during the 50-min perfusions. These results suggest that binding of parathion to BSA hinders its biotransformation by mouse livers perfused in situ, probably by limiting the availability of free pesticide to metabolic sites. Consequently, its elimination by the liver is insensitive to changes in hepatic blood flow. However, exclusion of BSA from the perfusate resulted in partitioning of all parathion in perfusate into the liver, leading to high concentrations of parathion within liver and preventing biotransformation of this pesticide.

Biotransformation of paraoxon and p-nitrophenol by isolated perfused mouse livers.

Single-pass perfusion in situ of mouse livers with the organophosphate paraoxon resulted in formation of p-nitrophenol (PNP), p-nitrophenyl sulfate (PNPS), and p-nitrophenyl-beta-D-glucuronide (PNPG). Following initiation of perfusion of paraoxon steady--state conditions were achieved in 15-25 min, at which time the extraction ratio was 0.55 (S.D. = 0.05). This suggests the capacity of mouse liver to biotransform paraoxon is not as great as previously reported. At all concentrations of paraoxon examined the amount of PNPS produced exceeded that of PNPG. However, as the concentration of paraoxon increased the relative proportion of PNP to PNPS and PNPG increased, indicating the capacity of liver to biotransform paraoxon exceeded the capacity to biotransform PNP. Single-pass perfusion in situ of mouse livers with PNP resulted in production of PNPS and PNPG. As with paraoxon, steady-state conditions were achieved in 15-25 min. The extraction ratio of PNP, as well as the metabolic profile, changed markedly with varying concentrations of PNP. At PNP reservoir concentrations of 4 microM or less the extraction ratio of PNP was 1, with all PNP metabolized to PNPS. As PNP concentrations increased (up to 75 microM) both unchanged PNP and PNPG appeared in the effluent. Thus the hepatic biotransformation of PNP was clearly dependent on substrate concentration.

Metabolic activation of phosphorothioate pesticides: role of the liver.

Mouse liver perfusion studies in situ revealed that the cholinesterase inhibitor chlorpyrifos oxon produced by the liver from the phosphorothioate pesticide chlorpyrifos was quickly detoxified within the liver, thereby preventing it's exit from the liver in the effluent. In contrast, when the pesticide parathion was perfused as a substrate a substantial amount of the toxic metabolite paraoxon was found in exiting perfusate. Pesticide concentrations (5-15 microM) used in the perfusion studies in situ were similar to their hepatic portal blood concentrations in vivo (2.32-12.95 microM) after i.p. administration of lethal or near lethal doses. Moreover, the half-life for elimination of paraoxon by mouse blood in vitro was 8.6 min, a rate sufficiently low to allow passage of paraoxon to extrahepatic target tissues from liver in vivo. These results suggest that in the mouse, the acute toxicity of chlorpyrifos is mediated by extrahepatic production of oxon, whereas that of parathion is likely mediated by both hepatic and extrahepatic activation.

Cholinergic drug effects and brain muscarinic receptor binding in aged rats.

Muscarinic systems are significantly altered in the brains of laboratory animals and man as a result of normal aging. Cholinergic neurotransmission in cerebral cortex and hippocampus is also severely impaired in a major age-related neurological disorder, Alzheimer's disease. The objective of these studies was to assess specific 3H-quinuclidinyl benzilate (3H-QNB) binding to brain muscarinic receptors in young, adult and senescent Fischer 344 rats, and to relate receptor changes to differences in the pharmacologic actions of cholinergic drugs. Muscarinic receptor density declined with advanced age in the frontal cortex, corpus striatum and hypothalamus, but no age-related changes in receptor affinity were observed. Specific binding of 3H-QNB in hippocampus was not significantly altered. In contrast, the in vivo effects of oxotremorine (hypothermia and antinociception) were markedly enhanced in aged rats, whereas scopolamine-induced locomotor activity was reduced. Hence, senescent rats were more sensitive to the pharmacologic actions of a cholinergic agonist, but less responsive than young rats to a muscarinic antagonist. These seemingly contradictory results of binding experiments and pharmacological studies could be due, in part, to changes in subtypes of brain muscarinic receptors with advanced age. Alternatively, the age-related differences in cholinergic drug effects may reflect a decreased ability of the senescent animal to adapt to changes in its environment.