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[(3)H]cimetidine, a radiolabeled histamine H(2) receptor antagonist, binds with high affinity to an unknown hemoprotein in the brain which is not the histamine H(2) receptor. Improgan, a close chemical congener of cimetidine, is a highly effective pain-relieving drug following CNS administration, yet its mechanism of action remains unknown. To test the hypothesis that the [(3)H]cimetidine-binding site is the improgan antinociceptive target, improgan, cimetidine, and 8 other chemical congeners were studied as potential inhibitors of [(3)H]cimetidine binding in membrane fractions from the rat brain. All compounds produced a concentration-dependent inhibition of [(3)H]cimetidine binding over a 500-fold range of potencies (K(i) values were 14.5 to >8000nM). However, antinociceptive potencies in rats did not significantly correlate with [(3)H]cimetidine-binding affinities (r=0.018, p=0.97, n=10). These results suggest that the [(3)H]cimetidine-binding site is not the analgesic target for improgan-like drugs.

Improgan, a cimetidine derivative which lacks activity at known histamine, opioid or cannabinoid receptors, acts by an unknown mechanism in the periaqueductal gray (PAG) and raphe magnus (RM) to stimulate descending, analgesic circuits. These circuits may utilize cannabinoid mechanisms. To characterize further the nature of these circuits, the effects of intracerebral (i.c.) microinjections of rimonabant (a CB(1) receptor inverse agonist) were studied on antinociceptive responses following i.c. microinjections of improgan and the cannabinoid agonist WIN 55,212 (WIN) in rats. Separate intra-RM injections of improgan (30 microg) and WIN (8 microg) produced near-maximal antinociception on both the hot plate (HP) and tail flick (TF) nociceptive tests. Pretreatment with intra-RM rimonabant (20 microg) antagonized the antinociception produced by both intra-RM improgan and intra-RM WIN, but had no effects when given alone. Similar studies with improgan demonstrated rimonabant-sensitive sites within the dorsal and ventrolateral PAG. However, intra-RM pretreatment with rimonabant had no effect on antinociceptive responses following intra-PAG improgan. These studies show that improgan activates pain-relieving mechanisms in the PAG and the RM, both of which may utilize local cannabinoid mechanisms.

[(3)H]Cimetidine (3HCIM) specifically binds to an unidentified site in the rat brain. Because recently described ligands for this site have pharmacological activity, 3HCIM binding was characterized. 3HCIM binding was saturable, heat-labile, and distinct from the histamine H(2) receptor. To test the hypothesis that 3HCIM binds to a cytochrome P450 (P450), the effects of nonselective and isoform-selective P450 inhibitors were studied. The heme inhibitor KCN and the nonselective P450 inhibitor metyrapone both produced complete, concentration-dependent inhibition of 3HCIM binding (K(i) = 1.3 mM and 11.9 muM, respectively). Binding was largely unaffected by inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2D6, 2E1, and 19A1 but was eliminated by inhibitors of CYP2C19 (tranylcypromine) and CYP3A4 (ketoconazole). Synthesis and testing of CC11 [4(5)-(benzylthiomethyl)-1H-imidazole] and CC12 [4(5)-((4-iodobenzyl)-thiomethyl)-1H-imidazole] confirmed both drugs to be high-affinity inhibitors of 3HCIM binding. On recombinant human P450s, CC12 was a potent inhibitor of CYP2B6 (IC(50) = 11.7 nM), CYP2C19 (51.4 nM), and CYP19A1 (140.7 nM) and had a range of activities (100-494 nM) on nine other isoforms. Although the 3HCIM binding site pharmacologically resembles some P450s, eight recombinant human P450s and three recombinant rat P450s did not exhibit 3HCIM binding. Inhibition by KCN and metyrapone suggests that 3HCIM binds to a heme-containing brain protein (possibly a P450). However, results with selective P450 inhibitors, recombinant P450 isoforms, and a P450 antibody did not identify a 3HCIM-binding P450 isoform. Finally, CC12 is a new, potent inhibitor of CYP2B6 and CYP2C19 that may be a valuable tool for P450 research.

Improgan is a congener of the H 2 antagonist cimetidine, which produces potent antinociception. Because a) the mechanism of action of improgan remains unknown and b) this drug may indirectly activate cannabinoid CB 1 receptors, the effects of the CB 1 antagonist/inverse agonist rimonabant (SR141716A) and 3 congeners with varying CB 1 potencies were studied on improgan antinociception after intracerebroventricular (icv) dosing in rats. Consistent with blockade of brain CB 1 receptors, rimonabant (K d = 0.23 nM), and O-1691 (K d = 0.22 nM) inhibited improgan antinociception by 48% and 70% after icv doses of 43 nmol and 25 nmol, respectively. However, 2 other derivatives with much lower CB 1 affinity (O-1876, K d = 139 nM and O-848, K d = 352 nM) unexpectedly blocked improgan antinociception by 65% and 50% after icv doses of 300 nmol and 30 nmol, respectively. These derivatives have 600-fold to 1500-fold lower CB 1 potencies than that of rimonabant, yet they retained improgan antagonist activity in vivo. In vitro dose-response curves with 35 S-GTPγS on CB 1 receptor-containing membranes confirmed the approximate relative potency of the derivatives at the CB 1 receptor. Although antagonism of improgan antinociception by rimonabant has previously implicated a mechanistic role for the CB 1 receptor, current findings with rimonabant congeners suggest that receptors other than, or in addition to CB 1 may participate in the pain-relieving mechanisms activated by this drug. The use of congeners such as O-848, which lack relevant CB 1 -blocking properties, will help to identify these cannabinoid-like, non-CB 1 mechanisms. Perspective This article describes new pharmacological characteristics of improgan, a pain-relieving drug that acts by an unknown mechanism. Improgan may use a marijuana-like (cannabinoid) pain-relieving mechanism, but it is shown presently that the principal cannabinoid receptor in the brain (CB 1 ) is not solely responsible for improgan analgesia.

Improgan, a chemical congener of cimetidine, is a highly effective non-opioid analgesic when injected into the CNS. Despite extensive characterization, neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. Presently, the specific binding of [(3)H]cimetidine (3HCIM) in brain fractions was used to discover 4(5)-((4-iodobenzyl)thiomethyl)-1H-imidazole, which behaved in vivo as the first improgan antagonist. The synthesis and pharmacological properties of this drug (named CC12) are described herein. In rats, CC12 (50-500nmol, i.c.v.) produced dose-dependent inhibition of improgan (200-400nmol) antinociception on the tail flick and hot plate tests. When given alone to rats, CC12 had no effects on nociceptive latencies, or on other observable behavioral or motor functions. Maximal inhibitory effects of CC12 (500nmol) were fully surmounted with a large i.c.v. dose of improgan (800nmol), demonstrating competitive antagonism. In mice, CC12 (200-400nmol, i.c.v.) behaved as a partial agonist, producing incomplete improgan antagonism, but also limited antinociception when given alone. Radioligand binding, receptor autoradiography, and electrophysiology experiments showed that CC12's antagonist properties are not explained by activity at 25 sites relevant to analgesia, including known receptors for cannabinoids, opioids or histamine. The use of CC12 as an improgan antagonist will facilitate the characterization of improgan analgesia. Furthermore, because CC12 was also found presently to inhibit opioid and cannabinoid antinociception, it is suggested that this drug modifies a biochemical mechanism shared by several classes of analgesics. Elucidation of this mechanism will enhance understanding of the biochemistry of pain relief.

Improgan is a chemical congener of the H2 antagonist cimetidine which shows the profile of a highly effective analgesic when administereddirectly into the CNS. Although the improgan receptor is unknown, improgan activates analgesic pathways which are independent of opioids, butmay utilize cannabinoid mechanisms. To discover selective, potent, improgan-like drugs, seven compounds chemically related to improgan weresynthesized and tested for antinociceptive activity in rats after intracerebroventricular (icv) administration. Among a series of improgan congenersin which the alkyl chain length of improgan ((eCH2)3e) was varied, five compounds showed full agonist antinociceptive activity withpotencies greater than that of improgan. VUF5420 (containing (eCH2)4e, EC50 ¼ 86.1 nmol) produced maximal antinociceptive activity afterdoses which showed no motor impairment or other obvious toxicity, and was 2.3-fold more potent than improgan (EC50 ¼ 199.5 nmol). Asfound previously with improgan, VUF5420-induced antinociception was unaffected by administration of the opioid antagonist naltrexone,but was inhibited by the CB1 antagonist SR141716A, suggesting a non-opioid, cannabinoid-related analgesic action. However, VUF5420showed very low affinity (Kd z 10 mM) on CB1-receptor activation of 35S-GTPgS binding, indicating that this drug does not directly interactwith the CB1 receptor in vivo. The present results show that VUF5420 is a high potency, improgan-like, non-opioid analgesic which mayindirectly activate cannabinoid pain-relieving mechanisms.

The antinociceptive profile of selected histamine H2 and histamine H3 receptor antagonists led to the discovery of improgan, a non-brainpenetrating analgesic agent which does not act on known histamine receptors. Because no chemical congener of improgan has yet been discovered which has both antinociceptive and brain-penetrating properties, the present study investigated the antinociceptive effects of a series of chemical compounds related to zolantidine, a brain-penetrating histamine H2 receptor antagonist. The drugs studied presently contain the piperidinomethylphenoxy (PMPO) moiety, hypothesized to introduce brain-penetrating characteristics. Following intracerebroventricular (i.c.v.) dosing in rats, six of eight drugs produced dose- and time-related antinociception on both the tail flick and hot plate tests over a nearly eight-fold range of potencies. Ataxia and other motor side effects were observed after high doses of these drugs, but two of the compounds (SKF94674 and loxtidine) produced maximal antinociception at doses which were completely devoid of these motor effects. Consistent with the hypothesis that PMPO-containing drugs are brain-penetrating analgesics, SKF94674 and another derivative (JB-9322) showed dose-dependent antinociceptive activity 15 to 30 min after systemic dosing in mice, but these effects were accompanied by seizures and death beginning 45 min after dosing. Other drugs showed a similar pattern of antinociceptive and toxic effects. In addition, loxtidine produced seizures without antinociception, whereas zolantidine produced neither effect after systemic dosing in mice. Although several of the drugs tested have histamine H2 receptor antagonist activity, neither the antinociception nor the toxicity was correlated with histamine H2 receptor activity. The present results are the first to demonstrate the existence of brain-penetrating antinociceptive agents chemically related to zolantidine and improgan, but further studies are needed to understand the mechanisms of both the pain relief and toxicity produced by these agents. © 2005 Elsevier B.V. All rights reserved.

Improgan, an analgesic derived from histamine antagonists, acts in the brain stem to activate descending non-opioid, pain-relieving circuits, but the mechanism of action of this drug remains elusive. Because improgan has a moderate affinity for 5-HT(3) receptors, and, since cholinergic and serotonergic drugs can modulate descending analgesic circuits, roles for 5-HT(3), nicotinic and muscarinic receptors in improgan antinociception were presently investigated in rats. Improgan (80 microg, icv) induced nearly maximal inhibition of hot plate and tail flick nociceptive responses, and these actions we unaffected by antagonists of muscarinic (atropine, 5.9 mg/kg, i.p.) and nicotinic (mecamylamine, 2 mg/kg, i.p.) receptors. Control experiments verified that these antagonist treatments were maximally effective against muscarinic and nicotinic antinociception in both tests. In addition, improgan antinociception was unaffected by icv pretreatment with a 5-HT(3) antagonist (ondansetron, 20 microg). When given alone, icv treatment with neither this antagonist nor a 5-HT(3) agonist (m-chlorophenylbiguanide, 1000 nmol, icv) modified thermal nociceptive latencies. These results show no role for supraspinal cholinergic and 5-HT(3) receptors in improgan antinociception. The findings help to narrow the search for the relevant mediators of the action of this novel analgesic agent.

Improgan is a compound developed from histamine antagonists which shows the pre-clinical profile of a highly effective, non-opioid analgesic when administered into the rodent CNS. Pharmacological studies suggest that improgan activates descending pain-relieving circuits, but the brain and spinal sites of action of this drug have not been previously studied. Presently, the effects of intracerebral and intrathecal microinjections of improgan were evaluated on thermal nociceptive responses in rats. Improgan produced large, dose- and time-related reductions in nociceptive responses following administration into the ventrolateral periaqueductal gray (PAG), the dorsal PAG, and the rostral ventromedial medulla (RVM). The drug had no measurable effects after injections into the caudate nucleus, basolateral amygdala, hippocampus, ventromedial hypothalamus, superior colliculi, ventrolateral medulla, or the spinal subarachnoid space. Inactivation of the RVM by muscimol microinjections completely attenuated antincociceptive responses produced by intraventricular improgan. These findings, taken with earlier results, show that, like opioids and cannabinoids, improgan acts in the PAG and RVM to activate descending analgesic systems. Unlike these other analgesics, improgan does not act in the spinal cord or in CNS areas outside of the brain stem. © 2004 Elsevier B.V. All rights reserved.

Histamine N-methyltransferase (HNMT), a cytosolic histamine-metabolizing enzyme, is the only known product of the 50-kb human HNMT. Here, a detailed investigation of HNMT products revealed the existence of a new brain mRNA product of HNMT. This species, named HNMT-Short (HNMT-S), encodes a 126-amino-acid protein. Northern blot analysis detected HNMT-S mRNA (1.0 kb) in placenta, but not in several other human tissues. In addition, unlike the known HNMT cDNA, HNMT-S cDNA did not result in histamine-methylating activity after transfection into COS-7 cells. These studies show that HNMT-S is a new mRNA species and putative protein product from HNMT. The physiological role of HNMT-S remains to be investigated.

Even though conventional systemic doses of cimetidine and other histamine H(2) antagonists display minimal brain penetration, central nervous system (CNS) effects (including seizures and analgesia) have been reported after administration of these drugs in animals and man. To test the hypothesis that cimetidine-like drugs produce these CNS effects via inhibition of GABA(A) receptors, the actions of these drugs were studied on seven different, precisely-defined rat recombinant GABA(A) receptors using whole-cell patch clamp recordings. The H(2) antagonists famotidine and tiotidine produced competitive and reversible inhibition of GABA-evoked currents in HEK293 cells transfected with various GABA(A) receptor subunits (IC(50) values were between 10-50 microM). In contrast, the H(2) antagonist ranitidine and the cimetidine congener improgan had very weak (if any) effects (IC(50) > 50 microM). Since the concentrations of cimetidine-like drugs required to inhibit GABA(A) receptors in vitro (greater than 50 microM) are considerably higher than those found during analgesia and/or seizures (1-2 microM), the present results suggest that cimetidine-like drugs do not appear to produce seizures or analgesia by directly inhibiting GABA(A) receptors.

In the CNS, histamine is a neurotransmitter that is inactivated by histamine N-methyltransferase (HNMT), a soluble enzyme localized to the cytosol of neurons and endothelial cells. However, it has not been established how extracellular histamine, a charged molecule at physiological pH, reaches intracellular HNMT. Present studies investigated two potential routes of histamine inactivation in mouse brain nerve terminal fractions (synaptosomes): (i) histamine uptake and (ii) histamine metabolism by HNMT. Intact synaptosomes demonstrated a weak temperature-dependent histamine uptake (0.098 pmol/min-mg protein), but contained a much greater capacity to metabolize histamine by HNMT (1.4 pmol/min-mg protein). Determination of the distribution of HNMT within synaptosomes revealed that synaptosomal membranes (devoid of soluble HNMT) contribute HNMT activity equivalent to intact synaptosomes (14.3 +/- 2.2 and 18.2 +/- 4.3 pmol/min-tube, respectively) and suggested that histamine-methylating activity is associated with the membrane fraction. Additional experimental findings that support this hypothesis include: (i) the histamine metabolite tele-methylhistamine (tMH) was found exclusively in the supernatant fraction following an HNMT assay with intact synaptosomes; (ii) the membrane-bound HNMT activity was shown to increase 6.5-fold upon the solubilization of the membranes with 0.1% Triton X-100; and (iii) HNMT activity from the S2 fraction, ruptured synaptosomes, and synaptosomal membranes displayed different stability profiles when stored over 23 days at - 20 degrees C. Taken together, these studies demonstrate functional evidence for the existence of membrane-bound HNMT. Although molecular studies have not yet identified the nature of this activity, the present work suggests that levels of biologically active histamine may be controlled by an extracellular process.

Breakdown of the blood brain barrier and the subsequent accumulation of free radicals, lactate, and glutamate appear to be the immediate causes of thiamine deficiency (TD)-induced damage to thalamus. The mechanisms triggering these events are unknown but recent evidence suggests an important role of histamine. We therefore studied the effects of histamine depletion on thalamic lesions in the pyrithiamine-induced thiamine deficient (PTD) rat. Chronic intracerebroventricular (i.c.v., 7 days) infusion of alpha-fluoromethylhistidine (FMH), combined with bilateral ibotenate destruction of the histamine-containing neurons in the tuberomammillary (TM) nucleus and bolus i.c.v. infusion of 48/80, a potent mast cell degranulating agent, was used to deplete brain histamine levels. PTD rats receiving combined FMH + 48/80 + TM lesions developed acute neurological symptoms, including spontaneous seizures, approximately 1 day earlier than PTD rats treated with i.c.v. infusion of vehicle and sham lesions of the TM. When examined 1 week after restoration of thiamine, the PTD vehicle + sham lesion animals contained severe neuronal loss and gliosis in midline, intralaminar, ventral, lateral, and posterior nuclei. PTD animals treated with FMH + 48/80 + TM lesions had little evidence of neuronal loss or microglial proliferation in thalamus except in the gelatinosus and anteroventral nuclei, in which there was complete neuronal loss. These data demonstrate a significant and regionally selective role of histamine in the development of thalamic lesions in a rat model of Wernicke's encephalopathy. Furthermore, these data suggest either a dissociation between seizures and thalamic lesions or a significant role of histamine in seizure-related damage to the thalamus.

Thioperamide, the prototypical histamine H(3) receptor antagonist, acts at the brain histamine H(3) autoreceptor to promote the release and metabolism of neuronal histamine, resulting in higher brain levels of the metabolite tele-methylhistamine. However, unlike thioperamide, several new histamine H(3) receptor antagonists enter the central nervous system (CNS), block brain histamine H(3) receptors and increase histamine release without increasing brain tele-methylhistamine levels. Experiments were performed presently in an attempt to understand these results. Consistent with previous findings, thioperamide significantly increased the content and synthesis rate of tele-methylhistamine in mouse and rat brain. In contrast, the histamine H(3) receptor antagonists GT-2227 (4-(6-cyclohexylhex-cis-3-enyl)imidazole) and clobenpropit did not affect tele-methylhistamine synthesis rate in mouse whole brain. The histamine H(3) receptor ligand GT-2016 (5-cyclohexyl-1-(4-imidazol-4-ylpiperidyl)pentan-1-one) had no effect on tele-methylhistamine levels in any rat brain region and decreased tele-methylhistamine synthesis rates in the mouse whole brain. To examine the possibility that these histamine H(3) receptor antagonists might prevent the methylation of newly released histamine, they were co-administered with thioperamide to determine their effects on the thioperamide-induced stimulation of tele-methylhistamine synthesis. GT-2016 significantly reduced the thioperamide-induced activation of tele-methylhistamine synthesis in mouse whole brain and in several regions of rat brain. Although further clarification is needed, these results suggest that some histamine H(3) receptor antagonists may promote the release of neuronal histamine, but also act to reduce histamine methylation in vivo by an unknown mechanism.

Improgan is the prototype drug from a new class of non-opioid analgesics chemically related to histamine and histamine antagonists, but the mechanism of action of these compounds has not been identified. Because several classes of analgesics act in the brain by reducing GABAergic inhibition of endogenous pain-relieving circuits, and because the activity of these substances is abolished by the GABA(A) agonist muscimol, the present study assessed the effects of muscimol on improgan antinociception in rats. Intracerebroventricular (icv) improgan (80 microg) and morphine (20 microg) both induced 80-100% of maximal analgesic responses on the tail flick test 10 to 30 min later. However, muscimol pretreatment (0.5 microg, icv) completely eliminated the antinociceptive activity of both compounds. Since improgan in vitro lacks activity at opioid and GABA(A) receptors, these findings: 1) confirm earlier literature showing that muscimol inhibits morphine analgesia, and 2) suggest that improgan activates a supraspinal, descending analgesic pathway, possibly via inhibition of GABAergic transmission. Since muscimol is the first compound discovered which inhibits improgan analgesia, muscimol will be a useful tool for the further characterization of this new class of pain-relieving substances.

Improgan is a derivative of cimetidine that induces non-opioid antinociception after intracerebroventricular (i.c.v.) administration, but the mechanism of action of this compound remains unknown. Since activation of either supraspinal or spinal alpha(2) adrenergic receptors can induce antinociception, and since improgan showed affinity for these receptors in vitro, the effects of the alpha(2) antagonist yohimbine on improgan antinociception were presently studied in rats on the hot plate and tail flick tests. Systemic yohimbine pretreatment (4 mg/kg, i.p.) completely blocked improgan antinociception (80 microg, i.c.v.), suggesting a mediator role for alpha(2) receptors. However, i.c.v. pretreatment with yohimbine (30 microg) had no effect on improgan antinociception. Since this treatment completely antagonized clonidine antinociception (40 microg, i.c.v.), supraspinal alpha(2) receptors seem to mediate the antinociceptive effects of clonidine, but not that produced by improgan. In contrast, intrathecal (i.t.) yohimbine pretreatment (30 microg) completely blocked the antinociception elicited by i.c.v. improgan and i.c.v. morphine. These results suggest that spinal (but not supraspinal) alpha(2) adrenergic receptors play a significant role in the pain-relieving actions of improgan. Furthermore, although improgan shows some affinity at alpha(2) receptors, this drug does not act directly at these receptors to induce antinociceptive responses. Like several other classes of analgesics, improgan-like drugs seem to activate non-opioid, descending pain-relieving circuits.

Abstract. The H 3 antagonist thioperamide is thought to act on brain H 3 autoreceptors to increase both the release and metabolism of neuronal histamine (HA). Our studies investigated the effects of several new brain-penetrating H 3 antagonists on rat cerebral cortical levels of the HA metabolite tele -methylhistamine ( t -MH). Animals were pretreated with H 3 antagonists (0.3 to 30 mg/kg; 1–4 hr; i.p.) in the presence or absence of the monoamine oxidase inhibitor pargyline to prevent metabolism of t -MH. Cortical t -MH levels were measured by both radioimmunoassay (RIA) and gas chromatography–mass spectrometry (GC–MS). Pargyline (60 mg/kg; 1 hr; i.p.) produced an ∼2-fold increase in t -MH levels as measured by either GC–MS or RIA. Thioperamide (± pargyline) increased t -MH levels as measured by both GC–MS and RIA. In contrast, neither 5-cyclohexyl-1-(4-imidazol-4-ylpiperidyl)pentan-1-one (GT-2016) (± pargyline), 4-(6-cyclohexylhex- cis -3enyl)imidazole (GT-2227) (± pargyline), nor clobenpropit (minus pargyline) increased t -MH levels as measured by GC–MS. A good agreement was found between t -MH levels as determined by either RIA or GC–MS except after treatment with GT-2016, which increased apparent t -MH brain levels according to the former but not the latter method. Subsequent studies suggest the in vivo formation of a GT-2016 metabolite, which can cross-react in the t -MH RIA. Although all H 3 receptor antagonists studied to date seem capable of enhancing brain HA release, only thioperamide presently was found to enhance cortical t- MH levels. Thus, H 3 receptor antagonists may differentially affect HA release and turnover, and brain t -MH levels may not be reliable predictors of H 3 agonist, partial agonist, or antagonist in vivo activity.

The current study examined the possible role of increased histamine release and granulocyte activity in the vascular changes that precede the onset of necrotic lesions with the thalamus of the pyrithiamine-induced thiamine deficiency (PTD) rat model of Wernicke's encephalopathy (WE). An increase in histamine release and the number of granulocytes was observed in lateral thalamus on day 9 and in medial thalamus on day 10 of PTD treatment, a duration of thiamine deficiency associated with perivascular edema in this brain region. Within the hippocampus, histamine release was significantly increased on day 9, declined to control levels on days 10-12, and was significantly elevated on days 12-14. No granulocytes were observed in hippocampus of either PTD or control rats. These observations suggest that the release of histamine from nerve terminals and histamine and other vasoactive substances from granulocytes may be responsible for thiamine deficiency-induced vascular breakdown and perivascular edema within thalamus.

Burimamide, a histamine (HA) derivative with both H2- and H3-blocking properties, induces antinociception when injected into the rodent CNS. Several related compounds share this property, and structure-activity studies have shown that this new class of analgesics is distinct from known HA antagonists. The prototype, named improgan, shows a preclinical profile of a highly effective analgesic, with activity against thermal, mechanical and inflammatory nociception after doses that do not alter motor balance or locomotor activity. Improgan analgesia is not blocked by opioid antagonists and is observed in opioid receptor knock-out mice. Unlike morphine, improgan does not induce tolerance after daily dosing. Extensive in vitro pharmacology studies have excluded known histaminergic, opioid, serotonergic, GABAergic and adrenergic receptor mechanisms, as well as 50 other sites of action. The improgan-like analgesic activity of some HA congeners suggests an analgesic action on a novel HA receptor, but further studies are required to substantiate this. Studies in progress are characterizing the sites and mechanisms of action of improgan, and developing brain-penetrating derivatives that could be useful for clinical pain.

Improgan is an analog of the H(2) antagonist cimetidine that does not act on known histamine receptors, but induces highly effective analgesia in rodents following intracerebroventricular (icv) administration. Since the mechanism of action of this compound remains unknown, improgan analgesia was characterized presently with the tail immersion nociceptive test in mutant mice lacking either the mu (exon 1 of MOR-1), delta (exon 2 of DOR-1) or kappa (exon 3 of KOR-1) opioid receptor. Improgan (30 microg, icv) induced reversible, maximal analgesia in both sexes of all three genotypes (+/+, +/- and -/-) of MOR-1 mutant mice 10 and 20 min after administration, whereas morphine analgesia was reduced (+/-) or abolished (-/-) in these subjects. In DOR-1 mutant mice, improgan was equally effective in all three genotypes, despite the reduction (+/-) or complete loss (-/-) of delta opioid receptor (3H-[D-Pen(2), D-Pen(5)]enkephalin, DPDPE) binding. Similarly, improgan analgesia was equivalent in all three genotypes of KOR-1 mutant mice, whereas kappa-mediated analgesia (U50,488) and kappa opioid (3H-U69,593) binding were abolished in the homozygous (-/-) mice. These studies demonstrate that improgan analgesia does not require intact MOR-1, DOR-1, or KOR-1 genes, and support the hypothesis that improgan-like analgesics act in the CNS by non-opioid mechanisms.

The effect of zolantidine dimaleate (ZOL), the first brain-penetrating histamine H2 receptor antagonist, was determined on morphine (MOR) antinociception (ANC) in rhesus monkeys. ZOL (0.75 mg/kg, s.c., given every 30 min), completely attenuated the ANC resulting from the lowest dose of MOR tested (1.0 mg/kg), with no effect on the responses to higher doses (3-10 mg/kg). ZOL had no effect on baseline nociceptive responses in the absence of MOR. Taken with previous studies on the pharmacological specificity of ZOL, the ANC properties of histamine, and more extensive studies in rodents, the present results suggest that opiates like MOR relieve pain in primates by mechanisms that include activation of brain H2 receptors.

The present study demonstrates that the putative antiaddictive agent ibogaine produces more robust behavioral effects in female than in male rats and that these behavioral differences correlate with higher levels of ibogaine in the brain and plasma of female rats. There were no differences in basal locomotor activity between the sexes, and the response of rats to ibogaine differed between the sexes even in the absence of morphine. Five h after receiving ibogaine (40 mg/kg, i.p.). antagonism of morphine-induced locomotor activity was evident in female but not in male rats. Either 19 h after administration of ibogaine (10-60 mg/kg, i.p.), or one h after administration of noribogaine (5-40 mg/kg, i.p.), a suspected metabolite, antagonism of morphine was significantly greater in female than in male rats. Brain and plasma levels of ibogaine (1 h) and noribogaine (5 h), measured by gas chromatography-mass spectrometry, were greater in females as compared with males receiving the same dose of ibogaine. Levels of both ibogaine and noribogaine were substantially lower at 19 h than at earlier times after ibogaine administration, contrary to a previous study in humans. For both sexes, subcutaneous administration of ibogaine (40 mg/kg, i.p., 19 h) produced greater antagonism of morphine-induced locomotor activity than did a comparable intraperitoneal injection, consistent with previous studies from this laboratory demonstrating that the former route of administration produces higher levels of ibogaine in the brain. These data show that there are sex differences in the effects of ibogaine and that this may be due to decreased bioavailability of ibogaine in males as compared to females.

Levels of histamine and its major metabolites in brain, tele-methylhistamine (t-MH) and tele-methylimidazoleacetic acid (t-MIAA), were measured in rat brains up to 12 h after intraperitoneal administration of L-histidine (His), the precursor of histamine. Compared with saline-treated controls, mean levels of histamine were elevated at 1 h (+102%) after a 500 mg/kg dose; levels of t-MH did not increase. Following a 1,000 mg/kg dose, mean histamine levels were increased for up to 7 h, peaked at 3 h, and returned to control levels within 12 h. In contrast, levels of t-MH showed a small increase only after 3 h; levels of t-MIAA remained unchanged after either dose. Failure of most newly formed histamine to undergo methylation, its major route of metabolism in brain, suggested that histamine was metabolized by another mechanism possibly following nonspecific decarboxylation. To test this hypothesis, other rats were injected with alpha-fluoromethylhistidine (alpha-FMHis; 75 mg/kg, i.p.), an irreversible inhibitor of specific histidine decarboxylase. Six hours after rats received alpha-FMHis, the mean brain histamine level was reduced 30% compared with saline-treated controls. Rats given His (1,000 mg/kg) 3 h after alpha-FMHis (75 mg/kg) and examined 3 h later had a higher (+112%) mean level of histamine than rats given alpha-FMHis, followed by saline. Levels of t-MH and t-MIAA did not increase. These results imply that high doses of His distort the simple precursor-product relationship between histamine and its methylated metabolites in brain. The possibility that some His may undergo nonspecific decarboxylation in brain after His loading is discussed. These findings, and other actions of His independent of histamine, raise questions about the validity of using His loading as a specific probe of brain histaminergic function.

Because histamine (HA) in the CNS may be a mediator of antinociception, a detailed investigation of the effects of the brain-penetrating H2 antagonist zolantidine (ZOL) was performed on five nociceptive tests in the presence and absence of morphine (MOR) in rats. ZOL inhibited MOR antinociception on the tail flick test, although a diurnal difference (inhibition in the dark cycle >> light cycle) was found. Similar results were found with the hot plate test, although details of the test procedure were significant. In contrast, ZOL induced opposing effects on MOR antinociception on two nonthermal tests (jump test and tail pinch test); ZOL alone induced moderate antinociception on the former test and mild antinociception on the latter test. Thus, ZOL exerts differential effects on baseline nociception and on MOR antinociception that vary depending on the nociceptive test employed, the light-dark cycle of the subjects, and the degree of stress associated with the nociceptive testing. These complex effects reveal the heterogeneous nature of opiate-induced modulation of nociception, and show that ZOL is a powerful tool for studying the relationships between opiates, HA, and nociceptive mechanisms.

A sensitive and highly selective analytical chemical method for measuring the indole alkaloid ibogaine in biological samples has been developed. The method utilizes organic extraction, derivatization with trifluoroacetic anhydride, and detection by combined gas chromatography-mass spectrometry. The deuterated analog of ibogaine, O-[C d 3 ]-ibogaine, was synthesized and used as an internal standard for the method. Standard curves, constructed from variable amounts of ibogaine (50–400 ng) and a fixed amount of internal standard (250 ng) were linear. The method has an approximate detection limit of at least 20ng/mL of tissue extract (180ng/g tissue), with a coefficient of variation of 8 to 12.5%. Chemical stability studies with the method found that aqueous ibogaine solutions (1–10 mg/mL) could be stored at 10 ° for up to 7 months with no more than 10% loss. The method was also used to measure brain ibogaine levels in rats 1 and 19 hr after a single dose of drug (40 mg/kg, i.p.); the results suggest a rapid disappearance of the drug after i.p. dosing. The method will help reveal the pharmacokinetic properties of this; putative anti-addictive agent in animals and humans.

The effects of the brain-penetrating H 2 antagonist zolantidine (ZOL, 3 mg/Kg, S.c.) were studied on morphine (MOR, 4 mg/Kg, S.c.) antinociception (tail flick test) in the presence and absence of previous restraint stress. Animals were handled for 3 days (to reduce handling stress), restrained for 1 hr or handled on day 4 and tested 24 hrs later. As found previously, restraint enhanced the intensity and duration of MOR antinociception. ZOL potentiated MOR antinociception in handled, non-restrained animals, but inhibited MOR action in restrained animals. In contrast, ZOL had no effects on nociceptive responses in either handled or stressed subjects in the absence of MOR. The data suggest that, in the absence of restraint, brain HA acts at the H 2 receptor to inhibit MOR antinociception. In contrast, when an animal has been previously restrained, HA enhances MOR antinociception. Thus, brain HA appears to mediate the restraintinduced potentiation of MOR antinociception. Taken with previous results, the present findings suggest that in the presence of MOR, brain HA can provide bidirectional modulation of nociception. The direction of the modulation seems to depend upon the stress experience of the animal.

Previous studies have shown that antinociceptive doses of systemic morphine increase extracellular histamine (HA) levels in the rat periaqueductal gray (PAG), although the cellular origin of basal and morphine-induced HA release in the PAG is unknown. Treatment with alpha-fluoromethylhistidine (FMH; 100 mg/kg, i.p.), the irreversible inhibitor of histidine decarboxylase, decreased basal HA release by a maximum of 80% and prevented morphine-induced HA release in the PAG. In addition, perfusion of this area with the sodium channel blocker tetrodotoxin (10(-6) M) decreased basal HA release by a maximum of 57% from baseline levels. When the perfusion medium was modified by substitution of magnesium for calcium, extracellular HA levels in the PAG decreased by a maximum of 72%, and morphine-induced HA release was prevented. Thioperamide (5 mg/kg, i.p.), an H3 antagonist, increased HA release in the PAG to a maximum of 249% within the first 30-60-min period. Taken together, these results suggest that basal and morphine-induced HA release in the rat PAG have a neuronal origin.

In the laboratory rat, inhalation (30 s) of high (70%) CO2 concentrations resulted in short-term (1-3 min) anesthesia, followed by a prolonged (up to 60 min) mild antinociception. Exposure to 100% CO2 resulted in significant thermal (hot-plate, 52 degrees, and tail-flick) and mechanical (tail-pinch, 886 g force) antinociception. Control animals, placed in the same chamber filled with air, showed no such effects. Rats exposed to 70% CO2 exhibited effects on the hot plate comparable to those seen after inhalation of 100% CO2, indicating that the response is not due to CO2-induced hypoxia. Additionally, recovery from halothane-induced anesthesia of comparable duration did not result in antinociception, confirming that anesthesia alone is not sufficient to produce the effect. Pretreatment with the opiate antagonist naltrexone (0.1-10 mg/kg i.p.) did not diminish the CO2-induced antinociception, suggesting that endogenous opioids are not obligatory in the mechanism of this response. Furthermore, hypophysectomy abolished hot-plate antinociception in animals exposed to 100% CO2 while sham-treated controls exhibited a pattern of hot-plate responses similar to that reported above. Taken together, these findings show that: (1) recovery from CO2-induced anesthesia results in a prolonged mild antinociception, detectable with thermal and mechanical nociceptive tests; and (2) this response may represent a novel from of environmentally induced antinociception, mediated by a non-opiate hormonal substance.

To determine the brain site of action of the H2 receptor antagonist tiotidine an inhibitor of systemic morphine (MOR) antinociception, the effects of intracerebral microinjections of this drug were studied on this response in rats. As assessed on the hot plate test, microinjections of tiotidine (1 ng in 0.5 μl) into the ventral lateral periaqueductal gray at the level of the dorsal raphe (PAG/DR) attenuated MOR-induced antinociceptive responses 10–15 min later, but potentiated these responses when tested 20–30 min after its administration. This treatment had neither effect in the absence of MOR. In contrast, intracerebral tiotidine had no effects on tail flick responses in the presence or absence of MOR. Intracerebral tiotidine reduced by about 50% the antinociception induced by systemic MOR (5.6 and 10 mg/kg), resulting in a parallel, rightward shift in the MOR dose-response curve. When testing occured with a fixed dose of MOR at a fixed time interval, tiotidine dose-response curves were U-shaped, an effect postulated to result from the separate inhibitory and stimulatory mechanism found at different times. Intracerebral mapping studies showed that the attenuation of MOR antinociception by tiotidine given into the PAG/DR was not reproduced by tiotidine injections into adjacent rostral, caudal, dorsal or ventral areas. Taken with previous studies showing that: (1) both MOR and histamine (HA) induce antinociception when given into the PAG/DR; and (2) systemic MOR releases HA in the PAG, the present results strongly suggest that systemic MOR attenuates supraspinally organized responses to phasic, thermal nociceptive stimuli by mechanism that include PAG HA release and subsequent activation of PAG H2 receptors. Furthermore, because H2 receptor antagonists given into the PAG/DR attenuate MOR antinociception as assessed on the hot plate, but not the tall flick text, these results provide further evidence for the dissociation of the mechanisms subserving MOR antinociception as assessed by these two texts.

The actions of zolantidine dimaleate and five other histamine H2 receptor antagonists, given into the lateral ventricle of rats, were assessed on nociceptive responses in the presence and absence of systemically administered morphine. On the tail flick response, zolantidine induced a time- and dose-dependent inhibition of morphine antinociception, with no effect on responses in the absence of morphine. Zolantidine and another H2 receptor antagonist, tiotidine, also inhibited morphine responses in the hot plate test. Four other H2 receptor antagonists of varying structure, brain-penetrating ability, and H2 potency also induced dose-related inhibition of morphine tail flick responses. Over three orders of magnitude, the potency of these compounds as inhibitors of morphine antinociception was highly correlated with H2 receptor antagonist potency (r = 0.98, P less than 0.005, n = 5). Taken with previous studies showing the selectivity of these compounds for histamine H2 receptors, and the antinociceptive properties of histamine, these results strongly suggest a role for brain histamine H2 receptors in the expression of morphine antinociception.

The effect of morphine on extracellular histamine levels in two regions of the rat midbrain was studied in vivo by microdialysis. Morphine (5.6 and 12.8 mg/kg, s.c.) significantly and dose-dependently increased extracellular histamine levels in the periaqueductal grey, while no significant effect was observed in the reticular formation. In addition, no significant effect of sequential saline injections was observed on extracellular histamine levels in the periaqueductal grey. Since morphine has no effect on histamine catabolism, these results suggest that morphine increases histamine release in the rat PAG, a site where morphine and histamine are known to have analgesic action. Taken with earlier studies showing the ability of H2 antagonists to block morphine analgesia, these results support the hypothesis that histamine and H2 receptors are important in mediating morphine analgesia in the rat periaqueductal grey. The cellular origin of the extracellular histamine, and the mechanism of this morphine effect remain to be determined.

A method for measuring histidine decarboxylase (HDC) in crude rat brain homogenates was developed by modification of existing 14 CO 2 -trapping methods. The addition of EDTA to tissue homogenates and assay buffer reduced non-enzymatic decarboxylation, and improved assay sensitivity and reliability. Addition of polyethylene glycol (molecular weight 300, PEG300) to the homogenizing buffer increased enzyme stability, permitting storage of crude homogenates. Studies of time course, tissue dilution and blanks showed that up to 8 mg of tissue could be assayed successfully with a 3.5-hr incubation. S - α -Fluoromethylhistidine (FMH) and α-hydrazinohistidine, specific inhibitors of HDC, induced concentration-dependent reductions of enzyme activity by up to 90%, whereas inhibitors of other decarboxylases had little or no effect. Kinetic studies of the enzyme in crude homogenates yielded K m and V max values similar to those found previously with other HDC methods, although a poor fit was found to a single enzyme model. When determined by the new method, the distribution of HDC in seven regions of the rat brain agreed well with previous results. The method is rapid, simple to perform, and requires no specialized equipment other than a scintillation counter.

The effects of morphine on the levels of histamine (HA), its metabolite tele-methylhistamine (t-MH) and on t-MH synthesis rates (thought to be indicative of neuronal HA release) were investigated in brain regions and spinal cords of DBA/2J (DBA) and C57/BL6 (C57) mice, two strains known to differ in their sensitivity to morphine. In DBA (a strain highly sensitive to morphine antinociception), morphine (10 mg/kg, s.c.) had no effect on brain regional t-MH or HA levels, but produced a generalized inhibition of regional t-MH synthesis rates ranging from 11 to 53%. The monoamine oxidase (MAO) inhibitor pargyline (used to estimate t-MH synthesis rates) had no effect on HA or t-MH levels in the DBA or C57 spinal cord, indicating the absence of detectable spinal HA turnover. Morphine (10 mg/kg) had no effect on DBA or C57 spinal cord HA or t-MH levels, but significantly increased t-MH synthesis in the DBA but not in the C57 spinal cord. These results suggest that in DBA mice, antinociceptive doses of morphine inhibit HA release in brain, and promote the release of HA from spinal cord. Neither effect was found in C57 mice, which are resistant to morphine antinociception. The relevance of these actions to previous studies showing the blockade of opiate antinociception by H2 antagonists remains to be established.

Histamine-synthesizing neurons in the brain may play an important role in cognition, and a histaminergic deficit has been found in Alzheimer's disease (AD). The AD medication tacrine was previously shown to inhibit some forms of rodent histamine-N-methyltransferase (HNMT), but the effects of AD drugs have not been investigated on human HNMT activity. Presently, the effects of tacrine and galanthamine (another AD medication) were studied on the activity of several forms of human and rat HNMT. Tacrine (0.01-10 microM) inhibited both human and rat HNMT activity in a concentration-dependent manner, but was less potent on both human embryonic kidney and recombinant human brain HNMT than on rat kidney HNMT (IC50 values were 0.46 and 0.70 microM vs. 0.29 microM, respectively). Galanthamine (up to 10 microM) did not influence the activity of rat kidney or human HNMT. Tacrine, but not galanthamine, may achieve brain levels sufficient to influence histamine metabolism in some patients treated for AD.

To investigate the pharmacokinetic properties of ibogaine, a putatively anti-addictive alkaloid, levels of this drug were quantified in plasma and tissues for up to 3 h following i.v. infusion in rats. Immediately following a 31-35 min infusion (20 mg/kg), mean plasma ibogaine levels were 373 ng/ml; these values declined rapidly thereafter in a biexponential manner. The plasma time course in 5 of 7 animals demonstrated an excellent fit to a two-compartment pharmacokinetic model, with alpha and beta half-lives of 7.3 min and 3.3 h, respectively. Drug clearance was estimated to be 5.9 l/h (n = 7). Ibogaine levels in brain, liver and kidney 3 h after the end of drug infusion were 143-170 ng/g, close to simulated values for the peripheral pharmacokinetic compartment. However, 3-h drug levels in adipose tissue were much higher (3,328 ng/g), implying the need for a more complex pharmacokinetic model. Mechanisms for the initial, rapid disappearance of plasma ibogaine are thought to include metabolic demethylation as well as redistribution to body stores. The sequestration of ibogaine by adipose tissue probably contributes to a protracted persistence of drug in the body. This persistence may be underestimated by the beta half-life reported in the present study.

Mast cells were positively identified in rat brain by a combination of staining and histochemical procedures. These cells stained positively with toluidine blue and Astrablau at low pH, indicating the presence of a proteoglycan similar to that found in peripheral mast cells. Brain mast cells also fluoresced after o-phthalaldehyde exposure, indicating that they contain histamine. Mast cells varied greatly in number among brains, but their distribution was almost exclusively thalamic; within thalamus, the ventral complex, medial dorsal, lateral, and paraventricular nuclei contained the most mast cells. Mast cell numbers were greater in brains of females than of males, and greater in left than in right hemispheres. These findings suggest that mast cells have a specialized function in thalamus and/or that the vascular environment of the thalamus is particularly conducive to mast cell accumulation.

A gas chromatographic-mass spectrometric method described by us to measure tele-methylhistamine (t-MH) in brain was used to measure t-MH in human cerebrospinal fluid (CSF), urine and plasma. The presence of t-MH in these body fluids was rigorously established. No pros-methyl-histamine could be detected, and it was used as internal standard to quantify t-MH in the fluids. The mean levels of t-MH were: urine, 943 pmol/mg creatinine; plasma, 12.3 pmol/ml; and CSF, 2.2 pmol/ml. Parallel measurements of histamine by a radioenzymatic method showed, respectively, 182 pmol/mg creatinine; 19.5 pmol/ml; and 388 pmol/ml. The levels of HA in CSF, much higher than those of its metabolite, t-MH, are high enough to stimulate HA receptors in the central nervous system.

Microwave irradiation focused on the head of small rodents is now widely used as a means of more accurately measuring acetylcholine, choline, cyclic AMP, and several other important brain constituents. Because of its probable neurotransmitter role and rapid turnover, a similar approach was taken to study brain histamine. Histamine was measured by a modified radio-enzymatic method and was found to be nearly tripled in brains from microwave treated rats, compared to decapitation controls (124 vs 42 ng/g). Possible explanations include a microwave-induced inactivation of histamine breakdown, a microwave-induced redistribution of previously unmeasured histamine, and microwave- induced histidine decarboxylation. Brain histamine remained unchanged up to 30 min after decapitation and microwave heated brains from decapitated rats also had elevated histamine levels, indicating that brain histamine levels in decapitated rats do not represent the remainder of a rapidly depleting pool. No evidence for previously unmeasured histamine was found. Furthermore, microwave irradiation did not enhance the formation of (3H)histamine after intraventricular C3H)histidine administration, indicating a lack of microwave-induced histidine decarboxylation. It is concluded that the elevation in rat brain histamine after focused microwave irradiation is probably not artifactual, although the mechanism responsible for this phenomenon remains obscure.

A role for brain histamine in the acute action of morphine was studied in mice. In contrast to earlier reports, whole brain histamine levels were not changed 30 min after morphine (1–56 mg/kg). Levels of the brain histamine metabolite, tele -methylhistamine, were also unchanged. Marphine (1.8–10 mg/kg) caused a dose-dependent antinociceptive response that was unaffected by histamine H1-antagonists (i.p.), H2-antagonists (i.p. or i.vent.), or metoprine (i.p.), an inhibitor of histamine metabolism. α-Fluoromethylhistidine, the inhibitor of brain histamine synthesis, unexpectedly potentiated the response to low doses of morphine. These results find no evidence for a role of brain histamine in opiate analgesia.

The effects of the opiate antagonist naloxone (10 mg/kg) and the histamine H2-antagonist cimetidine (100 mg/kg; both administered i.p.) were studied on the analgesia elicited by 3 currents of continuous-scrambled AC footshock (FSIA). Repeated analgesic measurements were made in each animal by use of the radiant heat tail-flick test. As shown by others, naloxone effectively inhibited the FSIA produced by 3 min of 2.0 mA, but had no effect on the responses elicited by higher currents (2.5 and 3.5 mA) of the same duration. Cimetidine significantly reduced the naloxone-insensitive FSIA after 3.5 mA, had no effect on that produced by 2.5 mA and potentiated the naloxone-sensitive analgesia elicited by 2.0 mA. These findings add to existing data supporting a role for brain histamine as a mediator of naloxone-insensitive analgesia, and also suggest the possibility that histamine may mediate hyperalgesic responses.

Rapid and complete inhibition of monoamine oxidase by pargyline produced linear increases in the content of the histamine metabolite, tele -methylhistamine (t-MH), in 9 regions of rat brain 2 and 4 h after drug administration. The treatment had little or no effect on the histamine content of these regions. As histamine methylation is the major metabolic pathway of histamine in brain, the rate of increase in brain t-MH content after complete inhibition of its metabolism provides an estimate of histamine turnover. Histamine turnover rates varied over 46-fold among regions, from cerebellum (0.029 nmol/g·h) to hypothalamus (1.33 nmolη), similar to those reported for norepinephrine and serotonin. Turnover rates were highly correlated with control t-MH levels ( r = 0.97) and control histamine levels ( r = 0.84). Rate constants were highest in the caudate nucleus and frontal cortex, equivalent to a half-life of about 11 min in these regions. While hypothalamic histamine had the highest turnover rate, the rate constant for histamine in this region was among the lowest in brain, perhaps consistent with the presence of histaminergic cell bodies. Histamine turnover rates may be indicative of the activity of histamine-synthesizing neurons, and their determination will facilitate understanding of histamine in brain.

The histamine metabolite tele-methylhistamine (t-MH) was identified and measured in crude and purified peritoneal mast cells (MCs). Peritoneal dialysates, peritoneal cells, and purified MCs all contained t-MH in concentrations representing about 0.2% of the corresponding histamine (HA) levels. T-MH levels in crude cells represented about 70% of the total dialysate levels, indicating the presence of extracellular as well as intracellular t-MH. T-MH levels per MC in purified fractions were similar to those of crude fractions, indicating a MC origin for the intracellular t-MH. Histamine methyltransferase activity was not detected in crude or purified MC fractions, and incubations with the monoamine oxidase inhibitor pargyline failed to increase the content or release of t-MH in either fraction, suggesting a very slow or non-existent histamine methylation in MCs. Compound 48/80 produced a temperature-dependent release of HA and t-MH in crude and purified preparations, and Triton X-100 also released both amines. In all cases, the degree of release of both amines was correlated, consistent with a granular origin for t-MH in MCs. The low concentrations of t-MH in MCs do not necessarily indicate a role for MCs in HA metabolism, but suggest that t-MH may be a valuable marker for non-MC HA.

The action of zolantidine, a histamine H2-receptor antagonist which penetrates the brain, was characterized on the analgesia elicited by brief continuous footshock of 3 intensities. Zolantidine inhibited the analgesia elicited by exposure to 3.5 mA for 3 min, with maximum inhibition observed 30 min after a dose of 5 mg/kg. This inhibition was not dose-related, possibly due to inhibition of the metabolism of histamine in brain by this drug at larger doses. While zolantidine alone, or in combination with naloxone, attenuated this analgesic response, naloxone alone had no such effect, further supporting the non-opiate nature of this response. Conversely, naloxone, but not zolantidine, strongly inhibited the analgesia elicited by 2.0 mA for 3 min, confirming the apparent opiate nature of this analgesic mechanism. Although a combination of zolantidine and naloxone still significantly attenuated this response, a tendency toward reversal of the antagonism by naloxone was observed with this combination of drugs, consistent with previous findings. Furthermore, the analgesia elicited by 2.5 mA for 3 min was resistant to both naloxone and zolantidine, alone or in combination. These results further strengthen the hypothesis that histamine and H2-receptors in brain are important mediators of endogenous antinociception and also support the possible existence of an additional non-opiate, non-H2-analgesic mechanism.

Antagonism of the H-2 receptor with cimetidine and other histaminergic receptor antagonists has been used to differentiate nonopioid and opioid forms of footshock analgesia which are mediated by neural mechanisms. Cimetidine reduces nonopioid footshock analgesia while potentiating an opioid form of this analgesia. The present study examined whether cimetidine altered the nonopioid, neurohormonal analgesia induced by either continuous cold-water swims (CCWS: 2 degrees C for 3.5 min) or the opioid analgesia induced by intermittent cold-water swims (ICWS: 2 degrees C, 18 10-sec swims, 18 10-sec recovery periods). Vehicle or cimetidine (10, 50, 100 mg/kg) injections were administered alone or paired with either CCWS or ICWS; tail-flick latencies, jump thresholds and core body temperatures were then measured. Cimetidine (100 mg/kg) significantly potentiated CCWS and ICWS analgesia and hypothermia, while having minimal effects upon basal thresholds. Lower cimetidine doses produced transitory effects on these measures. These data demonstrate dissociations between neural and neurohormonal forms of nonopioid analgesia following cimetidine treatment. The latter effect may be attributed to changes in stress responsiveness or thermoregulation rather than pain inhibition.

The effects of several variables related to incubation conditions were studied on both the basal and the histamine-stimulated 3H-cyclic AMP levels in a 3H-adenine prelabeled vesicular preparation of guinea pig cerebral cortex. Varying the preincubation time (i.e., the duration of incubation before labeling) caused pronounced differences in the postlabeling time course of 3H-cyclic AMP levels. A preincubation time of 45 min was found to minimize time-related postlabeling declines in 3H-cyclic AMP basal activity. Labeling of the homogenate for an entire experiment in one vessel ("bulk labeling") also contributed to time-related declines in activity; these changes were minimized by labeling aliquots of homogenate individually. Rapid sample mixing during incubation was found to be necessary to ensure adequate suspension of the preparation and to achieve a stable basal activity. Such mixing altered the pH of the samples; maintenance of the pH at 7.4 required a decrease in the bicarbonate concentration and/or use of supplementary buffers. Several parameters related to histamine concentration-response curves were found to be affected by various combinations of buffers and labeling methods. Individual labeling in Krebs-ringer-bicarbonate (15 mM) buffer gave the most stable and reproducible responses to histamine. This method permits detailed pharmacological characterization of transmitter-stimulated cyclic AMP changes in a cell-free system that retains many characteristics of brain slices.