Localization of cyclooxygenase-2 and prostaglandin E2 receptor EP3 in the rat lumbar spinal cord

Cyclooxygenase-2 (COX-2) is now considered to be the major constitutively expressed COX isozyme in the central nervous system. The present immunocytochemical study details localization of COX-2 immunoreactivity in rat spinal cord along with the expression of prostaglandin E2 receptor subtype EP3. Prominent COX-2 staining was observed in the nuclear envelope of neurons throughout the spinal cord, especially in the superficial dorsal horn laminae and motoneurons of lamina IX, as well as in glial cells of the white matter. Expression of EP3 receptor was strictly confined to afferent terminal areas in the superficial dorsal horns.     

NMDA receptor activation modulates evoked release of substance P from rat spinal cord

The possible modulation exerted by glutamate on substance P (SP) release from the rat spinal cord has been investigated. The N-methyl-D-aspartate (NMDA) receptor agonist, NMDA (1 microM), increased SP basal outflow by 46.5+/-10.9% (n = 3, P<0.01) without changing the evoked release of the peptide. Conversely, NMDA antagonists but not 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited both electrically-evoked and capsaicin-induced release of SP. In particular, D-2-amino-5-phosphonopentanoate (D-AP5; 50 microM) inhibited electrically-evoked and capsaicin-induced release of SP by 93+/-2.4% and 93.2+/-3.8% (n = 12, P<0.01), respectively. Functional pharmacological evidence is provided for glutamate exerting a positive feedback on SP release evoked by C fibre stimulation via NMDA receptor activation.     

Actions of prostaglandin E2 on rat supraoptic neurones

Prostaglandins (PGs) have been implicated in the regulation of vasopressin (VP) and oxytocin (OT) release in response to various stimuli. To examine the site and mechanism of actions of PGs, we studied effects of PGE2 and PG-receptor agonists on supraoptic nucleus (SON) neurones of rat hypothalamic slice preparations using extracellular recording and whole-cell patch-clamp techniques. PGE2 modulated the electrical activity of more than 80% of the neurones studied. The effects of PGE2 on both phasic and non-phasic neurones were mostly excitatory, and dose-dependent. The effects of PGE2 were mimicked by PGF2alpha or the FP agonist, fluprostenol, whereas PGD2 or the selective EP, IP or TP agonist was less effective or had no effect. The effects of PGE2 were unaffected by the EP1 antagonist, SC-51322, but reduced to 80% of control by the EP1/FP/TP antagonist, ONO-NT-012, which reduced the effects of fluprostenol to 32% of control. Moreover, some neurones responsive to PGE2 did not respond to fluprostenol. Patch-clamp analysis in SON slice preparations revealed that PGE2 at 10(-6) M depolarized the membrane potential by 3.9+/-0.3 mV from the resting membrane potential of -58.4+/-2.2 mV in the current-clamp mode. In the voltage-clamp mode, PGE2 induced inward currents at a holding potential of -70 or -80 mV, while it did not affect spontaneous excitatory postsynaptic currents. PGE2 induced currents also in dissociated SON neurones and the reversal potential of the currents was -35.5+/-0.9 mV, which was similar to that of currents induced by fluprostenol. These results suggest that SON neurones possess at least two types of PG receptors, FP receptors and EP receptors of a subclass different from EP1, EP2, or EP3, and that activation of these receptors leads to the opening of nonselective cation channels, membrane depolarization and increase of the action potential discharge.     

Macrophage-induced inhibition of nitric oxide production in primary rat hepatocyte cultures via prostaglandin E2 release

Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages. In cocultured hepatocytes incubated with LPS and IFN-gamma, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage-number-dependent. Macrophage-conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins. Ultrafiltration of macrophage-conditioned medium through a 500-d membrane to rule out higher-molecular-weight molecules, such as anti-inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophages did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by NG-monomethyl-L-arginine (L-NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E2 (PGE2) release by macrophages was concomitantly observed. Moreover, inhibition of PGE2 production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE2 release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.     

Transient spinal ischemia in rat: characterization of spinal cord blood flow, extracellular amino acid release, and concurrent histopathological damage

Extracellular concentrations of amino acids in halothane-anesthetized rats were measured using a microdialysis fiber inserted transversely through the dorsal spinal cord at the level of the lumbar enlargement in conjunction with HPLC and ultraviolet detection. After a 2-h washout and a 1-h control period, 20 min of reversible spinal cord ischemia was achieved by the inflation of a Fogarty F2 catheter passed through the femoral artery to the descending thoracic aorta. After 2 h of postischemic reperfusion, animals were transcardially perfused with saline followed by 10% formalin or 4% paraformaldehyde. The glutamate concentration in the dialysate was significantly elevated after 10 min of occlusion and returned to near-baseline during the first 30 min of reperfusion. Taurine was elevated significantly 0.5 h postocclusion and continued to increase throughout the 2 h of reperfusion. Glycine concentrations showed a tendency to be slightly above baseline during the reperfusion period. Glutamine concentrations modestly increased following 2 h of reperfusion. No significant changes in aspartate, asparagine, and serine were detected. In control animals no significant changes in any amino acids were detected. To assess the role of complete spinal ischemia on spinal glutamate release, studies were carried out using cardiac arrest. Twenty minutes after induction of cardiac arrest, the glutamate concentration was increased about 350-400%. In a separate group of animals, spinal cord blood flow (SCBF) and its response to decreased CO2 were measured using a laser probe implanted into the epidural space at the level of the L2 vertebral segment. SCBF decreased to 5-6% of the control during aortic occlusion. After reversible ischemia, marked hyperemia was seen for the first 15 min, followed by hypoperfusion at 60 min. Under control-preischemic conditions a decrease in arterial CO2 content caused a decrease in SCBF of about 25%. This autoregulatory response was almost completely absent when assessed 60 min after a 20-min interval of aortic occlusion. Histopathological analysis of spinal cord tissue from these animals demonstrated heavy neuronal argyrophilia affecting small and medium-sized neurons located predominantly in laminae III-V. These changes corresponded to signs of irreversible damage at the ultrastructural level. Occasionally, small areas of focal necrosis, located in the dorsolateral part of the dorsal horn and anterolateral part of the ventral horn, were found. The results are consistent with a role for glutamate in ischemically induced spinal cord damage and suggest that taurine elevation detected during the early reperfusion period may serve as an important indicator of irreversible spinal cord neuronal damage.     

Effect of bacterial endotoxin on the in vitro release of Type II phospholipase-A2 and prostaglandin E2 from human placenta

The aim of this study was to establish the effect of bacterial endotoxin lipopolysaccharide (LPS) on the release of Type II phospholipase-A2 (PLA2) and prostaglandin E2 (PGE2) from late-pregnant human placental tissue incubated in vitro. Under basal conditions, immunoreactive Type II PLA2 and PGE2 were released from tissue explants in a time-dependent manner (up to 24 h, ANOVA, P<0. 0001, n=6). The release of these mediators was not associated with a loss of cell membrane integrity, as indicated by concentrations of the intracellular enzyme, lactate dehydrogenase (LDH), in the incubation medium. Incubation of explants in the presence of LPS (0. 001-100 microg LPS/ml) significantly decreased PLA2 tissue content (P<0.02, n=6) and increased the accumulation of PLA2 and PGE2 in the incubation medium (P<0.0001, n=6). The data obtained in this study indicated that Type II PLA2 and PGE2 are released from term placenta under basal conditions and that LPS stimulated their release. The associated decrease in PLA2 tissue content is consistent with the hypothesis that LPS induces the release of stored PLA2. This study identifies one pathway by which products of bacterial infection may induce the release of a pro-inflammatory, extracellularly active PLA2 from intrauterine tissues that may promote the formation of phospholipid metabolites involved in the process of labour and delivery (e.g. the prostaglandins).     

Ethacrynic acid induced release of prostaglandin E to increase renal blood flow

Ethacrynic acid administered to anesthetized dogs was found to increase the level of prostaglandin E as determined by radioimmunoassay in renal venous blood at the time when renal blood flow was increased by this agent. No change was found in the renal venous level of prostaglandin F. When ethacrynic acid was administered after treatment with indomethacin, which blocks the increase in renal blood flow induced by the natriuretic agent, no increase in the renal venous level of prostaglandin E was seen. Thus, the dilation of the renal vasculature would appear to be caused by a stimulation of synthesis and release of prostaglandin E by ethacrynic acid.     

Protective effect of prostaglandin E2 on cerulein-induced rat pancreatitis]

In order to clarify the effect of prostaglandin E2 (PGE2) on cerulein-induced rat pancreatitis, we investigated the interaction of PGE2 with cerulein or secretin. Intravenous infusion of 10 micrograms/kg.h cerulein inhibited external secretion of the pancreas from one hour and caused macroscopic edema at 3 hours. Administration of PGE2 relieved the inhibitory effect of supramaximal dose of cerulein and decreased the pancreatic edema. The 100 micrograms/kg.hr PGE2 had no significant effect on the pancreatic juice volume and amylase secretion stimulated with 0.2 micrograms/kg.hr of cerulein. Intravenous injection of 100 micrograms/kg PGE2 inhibited both the volume and amylase secretion of pancreatic juice stimulated with 1 U/kg.h of secretin. The protective effect of PGE2 on cerulein-induced pancreatitis was not the stimulation on secretion but caused the cytoprotective effect of PG such as stabilization of cytoplasmic and lysosomal membrane.     

Spinal amino acid release and precipitated withdrawal in rats chronically infused with spinal morphine

Glutamate receptors are implicated in the genesis of opioid tolerance and dependence. Factors governing release of amino acids in systems chronically exposed to opiates, however, remain undefined. Using rats, each prepared with a spinal loop dialysis catheter and with a chronic lumbar intrathecal infusion catheter connected to a subcutaneous minipump, the release of amino acids before and during antagonist-precipitated withdrawal in unanesthetized rats was examined. Spinal infusion of morphine (20 nmol/micro l/hr) for 4 d had little effect on resting release of amino acids. In morphine-infused, but not saline-infused, rats naloxone (2 mg/kg, i.p.) evoked an immediate increase in the release of L-glutamate (299 +/- 143%) and taurine (306 +/- 113%) but not other amino acids. The magnitude and time course of the release of these amino acids significantly correlated with behavioral indices of withdrawal intensity. Acute intrathecal pretreatment immediately before naloxone with clonidine (20 microg; alpha2 agonist), MK-801 (3 microg; noncompetitive NMDA antagonist), or aminophosphonopentanoic acid (AP-5; 3 microg; competitive NMDA antagonist) suppressed naloxone-induced increases in spinal L-glutamate and taurine release and behavioral signs of withdrawal in spinal morphine-infused rats. Results point to a correlated increase in spinal L-glutamate release, which contributes to genesis of the opioid withdrawal syndrome. Agents such as clonidine that suppress opioid withdrawal may owe their action to an inhibition of excitatory amino acid release. The effects of MK-801 and AP-5 suggest a glutamate-evoked glutamate release.     

Protein kinase inhibitors attenuate cardiac swelling-induced amino acid release in the rat

Rat Langendorff heart preparations have been used to study the efflux of cardiac amino acids into coronary artery perfusates during brief (5-min) periods of exposure to hyposmotic stress (70 mM NaCl). Coronary flow rates, heart rates and intra-aortic pressures were recorded. Amino acid levels were measured by high-performance liquid chromatography. Hyposmotic stress caused marked percentage increases in taurine, glutamate and aspartate levels in the coronary perfusate, with smaller increases in phosphoethanolamine, glycine and alanine and non-significant increases in serine and glutamine. Amino acid levels declined during reperfusion with isosmotic Krebs-Henseleit bicarbonate buffer. Inhibition of protein kinase C with chelerythrine chloride (5 microM) depressed the osmotically-induced release of aspartate, glutamate, taurine and glycine. The protein tyrosine kinase inhibitor, genistein, reduced the anisosmotic efflux of aspartate, glutamate, taurine and phosphoethanolamine. Lavendustin A, another inhibitor of tyrosine kinase, depressed the osmotically evoked release of aspartate, glutamate and taurine. These studies demonstrate the involvement of protein kinase C and tyrosine kinases in the efflux of amino acids from the osmotically challenged rat heart and imply that these enzymes are involved in the mechanisms responsible for volume regulation by cardiac cells.     

Regulation of mouse bone marrow macrophage mannose receptor expression and activation by prostaglandin E and IFN-gamma

The macrophage mannose receptor mediates the clearance of microorganisms and glycoproteins containing terminal mannose oligosaccharides. Cell surface expression of this receptor progresses with macrophage differentiation, and thus may be critical to the scavenger function of tissue and circulating macrophages. Bone marrow macrophages, which were used in this study, differentiate in culture and express functional mannose receptors. The cytokine IFN-gamma triggered activation of these macrophages and down-regulated cell surface expression of the mannose receptor after 48 h. Macrophage activation, as assessed by the generation of superoxide radicals, was inversely correlated with mannose receptor expression. The number of surface receptors was diminished by exposure to IFN-gamma, whereas the binding affinity of the mannose receptor remained unchanged. Treatment with IFN-gamma reduced receptor biosynthesis yet did not alter receptor degradation. Mannose receptor biosynthesis is up-regulated by PG of the E series, and these anti-inflammatory agents reversed the effects of IFN-gamma on receptor expression. Down-regulation of the mannose receptor by IFN-gamma was fully reversible by PGE, indicating that receptor levels are dependent on the functional state of the cell rather than being linked to terminal cell differentiation. The regulation of the receptor by cytokines and anti-inflammatory reagents suggests that the mannose receptor plays a critical role in macrophage scavenger functions and potentially in modulating inflammatory reactions.     

The release of amino acids synthesised from various compartmented precursors in rat spinal cord slices

[14C]Glucose and [14C]acetate have been used to label amino acid pools believed to be localised in neurones and glia, respectively, in small slices of rat spinal cord. The effects of depolarising agents on the efflux of amino acids from these pools were compared and contrasted with their effect on the efflux of exogenous [3H]glutamate. Elevated (50 mM) potassium in the superfusing medium increased the release of glutamate, aspartate and GABA synthesised from either glucose or acetate and that of exogenous glutamate. These increases were not, however, abolished by tetrodotoxin (2 micron). Protoveratrine A (10(-4) M), on the other hand, elevated the efflux of glutamate, GABA and possibly aspartate when these amino acids were synthesised from glucose, but not when acetate was the labelled precursor. Furthermore, this effect was abolished by 2 micron tetrodotoxin. It is concluded that these techniques point to the existence in slices of spinal cord of neuronal pools of glutamate, GABA and possibly aspartate that may be released as a consequence of neuronal activity, and that these pools probably represent transmitter stores of these amino acids.     

Inhibition of prostaglandin E release by anti-inflammatory steroids

The aearance of tritium in plasma water was measured after i.p. injection of 2-tritio-L-alanine as an index of alanine transamination in rats. L-Cycloserine (10 mg/kg) and D-cycloserine (150 mg/kg) inhibited tritium release, whereas theophylline (100 mg/kg) stimulated tritium release.     

In vivo formation of prostaglandin E1 and prostaglandin E2 in atopic dermatitis

The authors present the young man case who had undergone 6 months long antibiotic therapy first, than has been operated because of growing nose obstruction with bloody pus secretion. Diagnostic difficulties were responsible for diagnosis and undertaking treatment. Fulminant course of disease resulted in death of patient in the course of immunosupression therapy.     

Inhibition of voltage-dependent calcium channels by prostaglandin E2 in rat melanotrophs

The effects of PGE2 on voltage-dependent Ca2+ channel currents were studied in dissociated rat melanotrophs by the whole-cell configuration of the patch-clamp technique. In about 90% of melanotrophs examined, PGE2 reversibly inhibited voltage-dependent Ba2+ currents elicited by voltage steps from a holding potential of -80 to 0 mV, with an ED50 of 68 nM. The maximum inhibition of Ba2+ currents by 1 microM PGE2 (35.3%) was comparable with that by the maximally effective concentration (100 nM) of dopamine. The EP1/EP3 PGE (EP) agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, mimicked the inhibition by PGE2, whereas the selective EP2 agonist, butaprostol, had little effect. The inhibition by PGE2 was partially, but significantly, reduced by the selective EP1 antagonist, SC-51322. The magnitude of the PGE2-induced inhibition of Ba2+ currents was greatly reduced by pretreatment with pertussis toxin, or by a depolarizing prepulse, to +80 mV, lasting for 50 msec. Although four distinct types (N-, P/Q-, L-, and R-types) of high-threshold Ba2+ currents were observed, PGE2 (1 microM) caused significant inhibition of only P/Q- and L-type currents, which were 17.3 and 10.1%, respectively, of the total Ba2+ currents. These results suggest that PGE2 inhibits P/Q- and L-type Ca2+ channels of rat melanotrophs via EP1 and EP3 receptors, which are coupled to pertussis toxin-sensitive G proteins, and produces both voltage-sensitive and -insensitive inhibition of Ca2+ channels.     

Prostaglandin E2 in human placenta: its vascular effects and activation of prostaglandin E2 formation by nicotine and cotinine

Tobacco smoking by pregnant women increases the frequency of spontaneous abortions and preterm births. Human labor is associated with enhanced intrauterine phospholipid metabolism and production of prostaglandin E2 (PGE2) which induces labor, initiates uterine contractions and maintains the homeostasis of placental blood flow. Therefore, we studied: (a) the influence of nicotine and cotinine on the effects of PGE2 on placental vasculature in perfused human placental cotyledon, and (b) the activation of placental phospholipase A2 (PLA2) by nicotine and cotinine using 1-palmitoyl-2-[1-14C]arachidonyl-phosphatidylethanolamine (PE, 2.2 nmol) as substrate. These studies revealed that: (1) increasing concentrations of PGE2 (10- 150 ng/ml) increased umbilical perfusion pressure by 170 +/- 10% (n = 6) of control (100%). Cotinine (2 microg/ml) enhanced this effect at all concentrations of PGE2. Nicotine (2 microg/ml) prevented the effect of PGE2; (2) both cotinine (EC50 470-500 fmol/l) and nicotine (EC50 18-32 pmol/l) activated PLA2 in human placental tissues. These observations indicated that cotinine was more potent than in nicotine activating PLA2 and potentiating the vasoconstrictive effects of PGE2 on fetal placental circulation. Nicotine activates nicotinic receptors and releases placental acetylcholine, a vasodilator of placental arteries. Acetylcholine stimulates muscarinic receptors of endothelial cells resulting in the release of endothelium-derived relaxing factor (EDRF), and possibly nitric oxide. Therefore, nicotine prevents or abolishes the vasoconstrictive effects of PGE2 through the release of EDRF. Cotinine is inactive at nicotinic and muscarinic receptors. Therefore, accumulation of cotinine, the major metabolite of nicotine, in fetal circulation may contribute to production of PGE2 and induction of preterm labor and spontaneous abortions.     

Involvement of L-type-like amino acid transporters in S-nitrosocysteine-stimulated noradrenaline release in the rat hippocampus

Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-L-cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [3H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na+-containing Tyrode's buffer, SNC-stimulated [3H]NA release was inhibited 30% by the coaddition of L-leucine. In the Na+-free, choline-containing buffer, SNC-stimulated [3H]NA release, which was similar to that in the Na+-containing buffer, was inhibited markedly by L-leucine, L-alanine, L-methionine, L-phenylalanine, and L-tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of L-leucine was stronger than that of D-leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [3H]NA release in the Na+-free buffer. Uptake of L-[3H]leucine into the slices in the Na+-free buffer was inhibited by SNC, BCH, and L-phenylalanine, but not by L-lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by L-leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 microM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na+-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Effect of 8-iso prostaglandin E2 on aqueous humor dynamics in monkeys

OBJECTIVE: To evaluate the effects of 8-iso prostaglandin E2 (8-iso PGE2; prosta-5,13-dien-1-oic acid,11,15-dihydroxy-9-oxo-,[5Z,8beta-11X,13E,15 S]-) on the intraocular pressure (IOP), outflow facility, and aqueous humor flow rates in normal monkeys and monkeys with glaucoma. METHODS: The IOP was measured before and as long as 6 hours after the topical application of 8-iso PGE2 to 1 eye of 6 normal monkeys and to the glaucomatous eye of 8 monkeys with unilateral laser-induced glaucoma. The pupil diameter was measured at the same times as the IOP measurements in the normal monkeys. Tonographic outflow facility and fluorophotometric flow rates of aqueous humor were measured in 6 normal monkeys before and after drug treatment. RESULTS: In normal monkeys, a single dose of 0.1% 8-iso PGE2 reduced (P<.01) the IOP for 4 hours in the treated eyes with a maximum (mean +/- SEM) reduction of 3.2 +/- 0.2 mm Hg, compared with the contralateral control eyes. The pupil size was smaller (P<.01) in the treated eyes by as much as 1.0 +/- 0.2 mm for 4 hours. In 8 glaucomatous monkey eyes, the application of 0.05% and 0.1% 8-iso PGE2 reduced the IOP (P<.01) for as long as 2 and 5 hours, respectively. The maximum reduction in the IOP was 4.6 +/- 0.8 mm Hg (0.05%) and 6.0 +/- 0.8 mm Hg (0.1%) compared with baseline measurements. The magnitude and duration of the ocular hypotensive effect were enhanced with twice-a-day administration for 5 consecutive days. Outflow facility in normal monkey eyes was increased (P<.05) by 48% in the treated eyes, and aqueous humor flow was unchanged (P>.10), compared with vehicle-treated contralateral control eyes. Mild eyelid edema, conjunctival edema, hyperemia, and discharge appeared in some eyes treated with the 0.1% drug concentration. CONCLUSIONS: The use of 8-iso PGE2 reduces the IOP in both normal and glaucomatous monkey eyes. An increase in outflow facility appears to account for most of the IOP reduction in normal monkeys. Clinical Relevance: The application of 8-iso PGE2 may have potential for the treatment of glaucoma as an outflow facility-increasing drug.     

Effect of continuous spinal remifentanil infusion on behaviour and spinal glutamate release evoked by subcutaneous formalin in the rat

Four mutants of Arabidopsis thaliana that are deficient in adenine phosphoribosyl transferase (APRT) activity have been isolated by selecting for germination of seeds and growth of the plantlets on a medium containing 2,6-diaminopurine (DAP), a toxic analog of adenine. In all mutants, DAP resistance is due to a recessive nuclear mutation at a locus designated apt. The mutants are male sterile due to pollen abortion after meiosis. Furthermore, it has been shown that metabolism of cytokinins is impaired in the mutant BM3, which has the lowest level of APRT activity among the mutants tested. However, three different cDNAs encoding APRT have been isolated in A. thaliana and this raised the question of the nature of the mutation which results in low APRT activity. The mutation was genetically mapped to chromosome I and lies within 6 cM of the phenotypic marker dis2, indicating that the mutation affects the APT1 gene, a result confirmed by sequencing of mutant alleles. The mutation in the allele apt1-3 is located at the 5' splicing site of the third intron, and eliminates a BstNI restriction site, as verified by Southern blotting and PCR fragment length analysis.