A Phase 1 Single-Ascending-Dose Trial in Healthy Volunteers to Evaluate the Safety, Tolerability, Pharmacokinetics, and Immunogenicity of Intravenous PNT001, a Novel Mid-domain Tau Antibody Targeting cis-pT231 Tau.

BACKGROUND: PNT001 is a humanized full-length IgG4 S228P monoclonal antibody that binds the cis conformation of the phosphorylated Thr231-Pro232 motif in human full-length (2N4R) tau (cis-pT231 tau) with high selectivity and affinity. It binds selectively to cis-pT231 tau in human tauopathy brain sections, inhibits aggregation of tau, and has shown efficacy in preclinical models of tauopathy. Good Laboratory Practice six-month toxicology studies in cynomolgous monkeys have shown no test article-related findings. OBJECTIVES: To evaluate the safety, tolerability, pharmacokinetics, and immunogenicity of single escalating intravenous doses of PNT001 in healthy volunteers. DESIGN: Phase 1, randomized, double-blind, and placebo-controlled 16-week study. SETTING: Subjects were recruited across three clinical research sites in the United States. PARTICIPANTS: Fifty healthy volunteer subjects enrolled, with 49 receiving the double-blind study drug. INTERVENTION: Six cohorts were administered single escalating doses of PNT001 (33, 100, 300, 900, 2,700, and 4,000 mg). The subjects were randomized 6:2 (PNT001:placebo). MEASUREMENTS: Safety was evaluated by the occurrence of adverse events, electrocardiography, physical examinations, neurological examinations, vital signs, and suicidality. Pharmacokinetics and biomarkers were assessed via serum and cerebrospinal fluid sample analyses. RESULTS: Dose continuation after review of sentinel group data and dose escalation after completion of full cohort data were determined by an external, independent safety board. There were no study pauses or safety concerns identified by the safety board. A total of 49 subjects received the study drugs, with 36 receiving PNT001 and 13 receiving placebo. There were three related non-serious adverse events, each Grade 1, which occurred at the lowest doses and resolved without sequelae. No maximum tolerated dose was identified, and no premature discontinuations, dose reductions, or interruptions due to treatment-related adverse events occurred. One unrelated serious adverse event occurred in a placebo subject with an undisclosed medical condition. No other safety findings were identified. Doses of 900-4,000 mg produced concentrations in the cerebrospinal fluid exceeding the binding affinity constant of PNT001 for cis-pT231 tau (45 ng/mL), indicating that concentrations sufficient for target engagement can be obtained in the cerebrospinal fluid within the tested dose range. The serum pharmacokinetic profile was as expected for a monoclonal antibody. The terminal half-lives ranged from 23.8-33.8 days, and the cerebrospinal fluid exposures were approximately 0.1% of the plasma concentration and dose-proportional. Of the 36 subjects receiving PNT001, one post-baseline positive anti-drug antibody result was observed at Day 112 in a subject who received PNT001 (300 mg). CONCLUSIONS: Single doses of PNT001 were safe and well-tolerated at all dose levels studied, including those doses expected to produce therapeutic benefit. These results support multiple ascending dose trials in patients with neurodegenerative tauopathies for this novel mid-domain tau antibody.

Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5.

Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3beta. Here we show that tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus/hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases.

Inhibition of neuronal calcium channels by a novel peptide spider toxin, DW13.3.

Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.

L-type calcium channels in insulin-secreting cells: biochemical characterization and phosphorylation in RINm5F cells.

Opening of dihydropyridine-sensitive voltage-dependent L-type Ca2+-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic beta-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[3H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that alpha1C subunits (199 kDa, short form) contribute only a minor portion of the total alpha1 immunoreactivity in membranes and partially purified Ca2+-channel preparations. However, alpha1C forms a major constituent of (+)-[3H]isradipine-labeled LTCCs as 54% of solubilized (+)-[3H]isradipine-binding activity was specifically immunoprecipitated by alpha1C antibodies. Phosphorylation of immunopurified alpha1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-beta-antibody directed against all known beta-subunits. Isoform-specific antibodies revealed that these mainly corresponded to beta1b- and beta3-subunits. We found beta2- and beta4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that alpha1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. beta1b- and beta3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.

Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits.

To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LC1 and LC2, and two size forms of the class D L-type alpha 1 subunit, LD1 and LD2. The larger isoforms had apparent molecular masses of approximately 200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5% acrylamide. Immunocytochemical studies using CNC1 and CND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.

Molecular cloning of the alpha-1 subunit of an omega-conotoxin-sensitive calcium channel.

Of the four major types of Ca channel described in vertebrate cells (designated T, L, N, and P), N-type Ca channels are unique in that they are found specifically in neurons, have been correlated with control of neurotransmitter release, and are blocked by omega-conotoxin, a neuropeptide toxin isolated from the marine snail Conus geographus. A set of overlapping cDNA clones were isolated and found to encode a Ca channel alpha-1 subunit, designated rbB-I. Polyclonal antiserum generated against a peptide from the rbB-I sequence selectively immunoprecipitates high-affinity 125I-labeled omega-conotoxin-binding sites from labeled rat forebrain membranes. PCR analysis shows that, like N-type Ca channels, expression of rbB-I is limited to the nervous system and neuronally derived cell lines. This brain Ca channel may mediate the omega-conotoxin-sensitive Ca influx required for neurotransmitter release at many synapses.

Phosphorylation of an alpha 1-like subunit of an omega-conotoxin-sensitive brain calcium channel by cAMP-dependent protein kinase and protein kinase.

Antibodies that recognize the alpha 2 delta and alpha 1 subunits of skeletal muscle L-type calcium channels have been used to investigate the subunit components and phosphorylation of omega-conotoxin (omega-CgTx)-sensitive N-type calcium channels from rabbit brain. Photolabeling of the N-type channel with a photoreactive derivative of 125I-omega-CgTx results in the identification of a single polypeptide of 240 kDa. MANC-1, a monoclonal antibody recognizing alpha 2 delta subunits of L-type calcium channels from skeletal muscle, immunoprecipitates the omega-CgTx-labeled 240-kDa polypeptide and approximately 6% of the digitonin-solubilized 125I-omega-CgTx-labeled N-type channels. MANC-1 also immunoprecipitates a phosphoprotein of 240 kDa that comigrates with 125I-omega-CgTx-labeled N-type calcium channels, but not with L-type calcium channels, in sucrose gradients. Both cAMP-dependent protein kinase and protein kinase C are effective in the phosphorylation of this polypeptide. Similar to the alpha 1 subunits of skeletal muscle L-type calcium channels, the immunoprecipitation of the 240-kDa phosphoprotein by MANC-1 is prevented by the detergent Triton X-100. Anti-CP-(1382-1400), an antipeptide antibody against a highly conserved segment of the alpha 1 subunits of calcium channels, immunoprecipitates the 240-kDa phosphopeptide in Triton X-100. The 240-kDa protein is phosphorylated to a stoichiometry of approximately 1 mol of phosphate/mol of omega-CgTx-binding N-type calcium channels by both cAMP-dependent protein kinase and protein kinase C. Our results show that the 240-kDa polypeptide is an alpha 1-like subunit of an omega-CgTx-sensitive N-type calcium channel. The N-type calcium channels containing this subunit are phosphorylated by cAMP-dependent protein kinase and protein kinase C and contain noncovalently associated alpha 1-like and alpha 2 delta-like subunits as part of their oligomeric structure.

Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons.

Integration and processing of electrical signals in individual neurons depend critically on the spatial distribution of ion channels on the cell surface. In hippocampal pyramidal neurons, voltage-sensitive calcium channels have important roles in the control of Ca2(+)-dependent cellular processes such as action potential generation, neurotransmitter release, and epileptogenesis. Long-term potentiation of synaptic transmission in the hippocampal pyramidal cell, a form of neuronal plasticity that is thought to represent a cellular correlate of learning and memory, is dependent on Ca2+ entry mediated by synaptic activation of glutamate receptors that have a high affinity for NMDA (N-methyl(-D-aspartate) and are located in distal dendrites. Stimuli causing long-term potentiation at these distal synapses also cause a large local increase in cytosolic Ca2+ in the proximal regions of dendrites. This increase has been proposed to result from activation of voltage-gated Ca2+ channels. At least four types of voltage-gated Ca2+ channels, designated N, L. T and P, may be involved in these processes. Here we show that L-type Ca2+ channels, visualized using a monoclonal antibody, are located in the cell bodies and proximal dendrites of hippocampal pyramidal cells and are clustered in high density at the base of major dendrites. We suggest that these high densities of L-type Ca2+ channels may serve to mediate Ca2+ entry into the pyramidal cell body and proximal dendrites in response to summed excitatory inputs to the distal dendrites and to initiate intracellular regulatory events in the cell body in response to the same synaptic inputs that cause long-term potentiation at distal dendritic synapses.

Subunit structure and localization of dihydropyridine-sensitive calcium channels in mammalian brain, spinal cord, and retina.

Monoclonal antibodies that recognize the alpha 2 delta subunits of calcium channels from skeletal muscle immunoprecipitate a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. They also immunoprecipitate 64% of rabbit brain dihydropyridine-sensitive calcium channels. Iodination of partially purified brain calcium channels followed by immunoprecipitation reveals alpha 1-, alpha 2 delta-, and beta-like subunits that have apparent molecular masses of 175, 142, and 57 kd, respectively. A polypeptide of 100 kd is also specifically immunoprecipitated. Immunocytochemical studies identify dihydropyridine-sensitive calcium channels in neuronal somata and proximal dendrites in rat brain, spinal cord, and retina. Staining of many neuronal somata is uneven, revealing relatively high densities of dihydropyridine-sensitive calcium channels at the base of major dendrites. L-type calcium channels in this location may serve to mediate long-lasting increases in intracellular calcium in the cell body in response to excitatory inputs to the dendrites.

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus.

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.

Calmodulin plays a dominant role in determining neurotransmitter regulation of neuronal adenylate cyclase.

Ca2+, through the mediation of calmodulin, stimulates the activity of brain adenylate cyclase. The growing awareness that fluctuating Ca2+ concentrations play a major role in intracellular signalling prompted the present study, which aimed to investigate the implications for neurotransmitter (receptor) regulation of enzymatic activity of this calmodulin regulation. The role of Ca2+/calmodulin in regulating neurotransmitter-mediated inhibition and stimulation was assessed in a number of rat brain areas. Ca2+/calmodulin stimulated adenylate cyclase activity in EGTA-washed plasma preparations from each region studied--from 1.3-fold (in striatum) to 3.4-fold (in cerebral cortex). The fold-stimulation produced by Ca2+/calmodulin was decreased in the presence of GTP, forskolin, or Mn2+. In EGTA-washed membranes, receptor-mediated inhibition of adenylate cyclase was strictly dependent upon Ca2+/calmodulin stimulation in all regions, except striatum. A requirement for Mg2+ in combination with Ca2+/calmodulin to observe neurotransmitter-mediated inhibition was also observed. In contrast, receptor-mediated stimulation of activity was much greater in the absence of Ca2+/calmodulin. The findings demonstrate that ambient Ca2+ concentrations, in concert with endogenous calmodulin, may play a central role in dictating whether inhibition or stimulation of adenylate cyclase by neurotransmitters may proceed.

Ca2+/calmodulin distinguishes between guanyl-5'-yl-imidodiphosphate- and opiate-mediated inhibition of rat striatal adenylate cyclase.

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Calmodulin may play a pivotal role in neurotransmitter-mediated inhibition and stimulation of rat cerebellar adenylate cyclase.

Adenylate cyclase activity was stimulated 2.5-fold by exogenous Ca2+/calmodulin (CaM) (1 microM) in rat cerebellar plasma membranes which had been depleted of endogenous Ca2+/CaM. In EGTA-washed membranes, phenylisopropyladenosine (an adenosine receptor agonist) was unable to inhibit adenylate cyclase activity unless exogenous Ca2+/CaM was included in the assay. Conversely, isoproterenol (a beta-adrenergic receptor agonist) was able to stimulate adenylate cyclase activity only in the absence of Ca2+/CaM. The regulation of adenylate cyclase activity by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p, a nonhydrolyzable guanine nucleotide analog, used to monitor interactions between guanine nucleotide-binding proteins and the catalytic unit of adenylate cyclase] was similar to that of phenylisopropyladenosine and isoproterenol; i.e., Gpp(NH)p produced inhibition exclusively in the presence of Ca2+/CaM, whereas only stimulation was observed in the absence of Ca2+/CaM. These results suggest that changes in intracellular Ca2+ concentrations may determine whether adenylate cyclase can be stimulated or inhibited by neurotransmitters.

Antagonism of calmodulin-stimulated adenylate cyclase by trifluoperazine, calmidazolium and W-7 in rat cerebellar membranes.

Ca++-calmodulin (CaM)-dependent stimulation of adenylate cyclase in rat cerebellar plasma membranes was demonstrated by removal of endogenous Ca++ and CaM and addition of exogenous Ca++ and CaM to the membranes. This CaM-dependent adenylate cyclase activity could be inhibited by calmidazolium and trifluoperazine in an apparently competitive manner, whereas the inhibition produced by W-7 was not competitive. The potency of the antagonists was strictly dependent upon the concentration of exogenous CaM present in the assay. Preincubation of membranes with exogenous CaM, followed by addition of anti-CaM agents, greatly reduced the inhibition of CaM-dependent adenylate cyclase activity. The potency of the anti-CaM agents was further decreased in membranes that had not been depleted of endogenous Ca++ and CaM (native membranes). The results suggest that optimal inhibition of CaM-dependent adenylate cyclase activity occurs upon simultaneous addition of exogenous CaM and anti-CaM agents to membranes depleted of endogenous Ca++ and CaM. Association of CaM with the catalytic unit of adenylate cyclase before introduction of CaM antagonists results in a CaM-C complex that is relatively refractory to inhibition by these anti-CaM agents. This CaM-catalytic unit complex probably exists in native membranes, rendering the antagonism of adenylate cyclase activity supported by endogenous Ca++-CaM essentially insensitive to low concentrations of CaM antagonists.

Changes in adenosine receptor sensitivity in morphine-tolerant and -dependent mice.

Treatment of mice with either a 75-mg morphine pellet (72 h) s.c. or 100 mg/kg of morphine s.c. (3.5 h) did not alter the ED50 of (-)-N6-(phenylisopropyl)adenosine (PIA) in the tail-flick assay. Under the same treatment conditions, caffeine became a more potent antagonist of PIA-induced analgesia, and the dose-response curve for the locomotor effects of caffeine was shifted to the left. There was no change from control in the distribution of caffeine to the brain in mice pretreated with morphine. Brain levels of PIA were decreased significantly at the two highest doses used to elicit analgesia in morphine-implanted mice when compared to control. Adenosine receptor binding assays, utilizing [3H]PIA and [3H]diethylphenylxanthine as agonist and antagonist ligands, respectively, revealed significant increases in the Bmax values for both ligands without changes in Kd in morphine-implanted mice when compared to control. These data suggest that there is an increase in sensitivity to drugs which interact with adenosine receptors in morphine-tolerant and -dependent mice.

Dopamine, acting through D-2 receptors, inhibits rat striatal adenylate cyclase by a GTP-dependent process.

This report demonstrates that the D-2 dopamine receptors that are present in rat striatum can directly inhibit the activity of adenylate cyclase in a GTP-dependent manner. N-n-propylnorapomorphine evoked a more pronounced inhibition than did dopamine. However, in the presence of the D-1-selective antagonist, SCH 23390, dopamine was also observed to inhibit the enzyme. Forskolin facilitated the detection of D-2 receptor-mediated inhibition by markedly stimulating striatal adenylate cyclase activity. The inhibition was antagonized in a dose-dependent manner by the D-2 receptor antagonist spiperone (Ki value = 70 pM) and was absolutely dependent on the presence of both GTP and sodium ions. Inhibition produced via D-2 receptors was additive with that produced via opiate or adenosine A1 receptors. The nonhydrolyzable GTP analogue, 5'-guanylylimidodiphosphate [Gpp(NH)p], did not substitute for GTP in promoting the D-2 receptor-mediated inhibition. It thus appears that D-2 receptors mediate adenylate cyclase inhibition by processes that have been observed for other neurotransmitters in the striatum and elsewhere. In addition, Gpp(NH)p displayed a Ca2+/calmodulin dependency for its inhibitory effects that was not shared by receptor-mediated, GTP-dependent inhibition.

Cross-tolerance studies between caffeine and (-)-N6-(phenylisopropyl)-adenosine (PIA) in mice.

Chronic administration of caffeine to mice (1 mg/ml in drinking water X 14 d) led to a downward shift in the dose-response curve for the locomotor effects of caffeine. Caffeine was also less effective as an antagonist against (-)-(N6-phenylisopropyl)-adenosine (PIA)-induced analgesia in the tail flick assay in these animals. The dose-response curves of PIA for both analgesia and locomotor depression were shifted to the left in animals chronically administered caffeine. In mice chronically administered PIA (1 mg/kg/d X 14 d), the dose-response curves of PIA for both analgesia and locomotor depression were shifted to the right. The dose-response curve for the locomotor effects of caffeine was shifted to the left, and caffeine exhibited greater antagonist activity against the analgesic action of PIA in these animals. There was no change in the Kd or Bmax values of either 3H-PIA or 3H-diethylphenylxanthine (DPX, a potent adenosine receptor antagonist) in mice chronically administered PIA. The Bmax values for both 3H-PIA and 3H-DPX were significantly increased, while the Kd values were not changed in mice chronically administered caffeine. There was no detectable change in the brain levels of either PIA or caffeine in animals chronically treated with either drug. The results demonstrate that chronic administration of caffeine increases the sensitivity of mice to the actions of PIA and vice versa, providing supportive evidence for the interaction of these drugs at the same receptor, which is probably an adenosine receptor.

The effect of chronic administration of caffeine on morphine-induced analgesia, tolerance and dependence in mice.

Morphine-induced analgesia, and the development of morphine-induced tolerance and dependence was determined in mice which had drunk caffeinated water (1 mg/ml) for 14 days or in mice which had received (-)-N6-(phenylisopropyl)-adenosine (PIA) 1 mg/kg i.p. for 14 days. Analgesia was assessed by the tail flick assay. The development of dependence was assessed by determining the ED50 of naloxone to precipitate withdrawal jumping (3 h after 100 mg/kg morphine pretreatment or 72 h after s.c. implantation of a morphine 75 mg pellet) and by determining the extent of naloxone-precipitated hypothermia in morphine-implanted animals. In mice chronically administered caffeine, the ED50 for morphine-induced analgesia was significantly decreased while the naloxone ED50 for withdrawal jumping increased by 2-fold after both types of morphine pretreatment. In control animals (tap water for 14 days), doses of 1 and 10 mg/kg of naloxone caused significant hypothermia in morphine-implanted animals. Doses of naloxone up to 100 mg/kg did not cause significant hypothermia in morphine-implanted animals which had received chronic caffeine. The development of tolerance was determined by computing the morphine potency ratio for the tail flick assay (tolerant ED50/control ED50). In mice chronically administered caffeine, the potency ratio was decreased significantly in morphine-implanted animals when compared to control. Morphine-induced analgesia, tolerance and dependence was not changed significantly in animals chronically administered PIA. Neither the distribution of morphine to the brain nor the opioid receptor binding parameters for [3H]etorphine and [3H]naltrexone were altered in mice chronically administered caffeine or PIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of caffeine and 8-phenyltheophylline on the actions of purines and opiates in the guinea-pig ileum.

The antagonist effects of 8-phenyltheophylline (8-PT) and caffeine against the actions of adenosine, (-)-N6-(R-phenyl-isopropyl)-adenosine (PIA), morphine and nalorphine on the guinea pig ileum preparation were examined. Antagonism of both adenosine and PIA by caffeine and 8-PT was concentration dependent. The slopes of Schild plots for both alkylxanthines vs. adenosine were significantly less than -1 unless the adenosine reuptake blocker dipyridamole (0.1 microM) was include in the tissue bath. Under these conditions, the 95% confidence intervals of the Schild plot slopes embraced theoretical unity, suggesting competitive antagonism. The antagonism of PIA by the alkylxanthine was also competitive. Dipyridamole had no effect on the potency of PIA. The pA2 value for caffeine-adenosine was not different from that for caffeine-PIA, and the pA2 values of 8-PT-adenosine and 8-PT-PIA were similar, suggesting that these two agonists interacted with similar receptors. The pA2 values using 8-PT were approximately 1.5-fold higher than those employing caffeine, suggesting higher affinity for 8-PT at these receptors. Caffeine significantly antagonized morphine at all concentrations used (0.5-1.0 mM), but only antagonized nalorphine at the two highest concentrations. After treating ilea with the mu-specific irreversible antagonist beta-FNA (beta-funaltrexamine) (resulting in a preparation with relatively pure kappa receptor population), the antagonist effect of caffeine against morphine was reduced such that only a concentration of 1 mM resulted in significant antagonism, while the effects of caffeine against nalorphine were unchanged. 8-PT did not antagonize morphine or nalorphine.(ABSTRACT TRUNCATED AT 250 WORDS)