Minimally invasive removal of a recurrent lumbar herniated nucleus pulposus by the small incised microendoscopic discectomy interlaminar approach.

In this report, we introduce two cases of recurrent herniated nucleus pulposus (HNP) at L5-S1 that were successfully removed using the small incised microendoscopic discectomy (sMED) technique, proposed by Dezawa and Sairyo in 2011. sMED was performed via the interlaminar approach with a percutaneous endoscope. The patients had previously underdone microendoscopic discectomy for HNP. For the recurrent HNP, the sMED interlaminar approach was selected because the HNP occurred at the level of L5-S1; the percutaneous endoscopic transforaminal approach was not possible for anatomical reasons. To perform sMED via the interlaminar approach, we employed new, specially made devices to enable us to use this technique. In conclusion, sMED is the most minimally invasive approach available for HNP, and its limitations have been gradually eliminated with the introduction specially made devices. In the near future, percutaneous endoscopic surgery could be the gold standard for minimally invasive disc surgery.

Endoscopic removal of herniated nucleus pulposus migrated dorsally to the dural sac.

A herniated nucleus pulposus (HNP) migrated dorsally to the dural sac is a rare condition. Here, we present a case, in which the HNP was removed with minimally invasive spinal endoscopy. A 54-year-old man presented complaining of left leg pain and paresis. Neurologic findings and an MRI suggested an epidural tumor or a dorsally migrated HNP compressing the S1 nerve root and dural sac. With a spinal endoscope, careful laminotomy of caudal L5 and cranial S1 was made. En bloc flavectomy exposed a mass covered with a thin capsule. The mass was identified as a dorsally migrated HNP. After complete HNP fragment removal, the dural sac and S1 nerve root were decompressed. Immediately postoperative, the leg pain subsided and motor function normalized, although the patient complained of numbness at the S1 dermatome area. In summary, a large HNP that had migrated dorsally to the dural sac was successfully removed endoscopically.

Hypoalgesic behaviors of P/Q-type voltage-gated Ca2+ channel mutant mouse, rolling mouse Nagoya.

Rolling mouse Nagoya (tg(rol)) is a spontaneously occurring P/Q-type voltage-gated Ca2+ channel (VGCC) mutant mouse. A P/Q-type VGCC with the tg(rol) mutation has lower voltage sensitivity of activation, and mice with a homozygous genotype (tg(rol)/tg(rol)) but not with a heterozygous genotype (tg(rol)/+) show impaired motor coordination of the hind limbs. To investigate the roles of P/Q-type VGCC in pain sensing mechanisms, behavioral responses of adult tg(rol) mice to thermal, mechanical and chemical nociceptive stimuli were examined by the plantar, tail-flick, von Frey and formalin tests. The latency of the withdrawal response to thermal stimuli in the plantar or tail-flick tests was significantly longer in tg(rol)/tg(rol) mice than in tg(rol)/+ and wild-type (+/+) mice, and in tg(rol)/+ mice than in +/+ mice. The withdrawal response to mechanical stimuli in the von Frey test was lower in tg(rol)/tg(rol) mice than in +/+ mice. Although the licking time during the first 5 min after the formalin injection was similar among all of the three genotypes, that during 5-60 min was significantly shorter in tg(rol)/tg(rol) mice than in tg(rol)/+ and +/+ mice, and in tg(rol)/+ mice than in +/+ mice. Artificial inflammation induced by injection of complete Freund's adjuvant (CFA) into a hind paw significantly enhanced the withdrawal response recorded in the plantar and von Frey tests regardless of the mouse genotype. The CFA-enhanced response in the tg(rol)/tg(rol) mice was similar to the response in +/+ mice without the CFA injection. These results suggest that tg(rol) mutant mice show hypoalgesic responses caused by a lower sensitivity to nociceptive thermal, mechanical and chemical stimuli. It is concluded that the P/Q-type VGCC has a pro-nociceptive role and that the tg(rol) mutant mouse may be a useful tool to investigate the role of the P/Q-type VGCC in pain sensing mechanisms.

The role of calcium in the action and release of vasopressin and oxytocin from CNS neurones/terminals to the heart.

The main long-range goal of this study is to analyse how electrical activity generated at somata is transformed into chemical signals at nerve terminals. We try to achieve this goal by examining, at the level of membrane and molecular mechanisms, the steps considered to be involved in stimulus-secretion coupling: how neurotransmitters are released in response to depolarisation of the nerve terminal membrane. We have demonstrated over several years the release of the neuroactive peptides, vasopressin and oxytocin and the role for Ca(2+), in the hypothalamic-neurohypophysial system (HNS) of the rat and also in cardiac tissues (from the brain to the heart). This study was performed using both a well-characterized preparation of pure, isolated neurohypophysial nerve terminals, a preparation of isolated hypothalamic magnocellular neurones and isolated cardiac myocytes. Furthermore, the intact HNS would affords the unique opportunity of comparing the somata and terminals of the same CNS neurones. This article plans to build on the this wealth of information already gathered on isolated, individual terminals/somata in order to analysis of the physiology of the whole, intact system in situ. We show some of the well established data to explain: i) why are different patterns of electrical activity (i.e. bursts) best for AVP vs. OT release in the intact HNS, ii) are there any other parameters, transmitters, messengers, hormones and drugs that could play an important role, iii) is Ca(2+) important to understand this physiology, and finally iv) what do we learn from the comparison to the cardiac system?

Acute estradiol application increases inward and decreases outward whole-cell currents of neurons in rat hypothalamic ventromedial nucleus.

Acute estradiol (E2) can potentiate the excitatory responses of hypothalamic ventromedial nucleus (VMN) neurons to neurotransmitters. To investigate the mechanism(s) underlying the potentiation, the whole-cell patch voltage clamp technique was used to study VMN neurons in hypothalamic slices prepared from female juvenile (3-5 weeks) rats. A voltage step and/or ramp was applied every 5 min to evoke whole-cell currents before, during and after a treatment with E2 (10 nM), corticosterone (10 nM) or vehicle for up to 20 min. Acute E2 increased inward currents in 38% of neurons tested. Their average peak inward current amplitudes started to increase within 5 min and reached the maximum of 163% of pretreatment level (Pre) at 20 min of treatment before recovering toward Pre. These increases are significantly greater than the Pre and corresponding vehicle controls and non-responsive neurons. Outward currents were decreased significantly by E2 in 27% of E2-treated cells, down to 60% of Pre levels. E2 also appeared to affect the kinetics of the inward and outward currents of estrogen-responsive neurons. Whenever observed, the effects of acute E2 were reversible after a 5- to 10-min washing. Probability analysis indicates that E2 affected the inward and the outward currents independently. The E2 effects are specific in that they were not produced by similar treatment with vehicle or corticosterone. Pharmacological characterizations using ion replacement and channel blockers showed that the inward currents were mediated practically all by Na(+) and the outward currents mainly by K(+). Thus, acute E2 can enhance inward Na(+) and attenuate outward K(+) currents. Since both effects will lead to an increase in neuronal excitability, they may explain our previous observation that E2 potentiates the excitation of VMN neurons.

Intracellular calcium increase and somatodendritic vasopressin release by vasopressin receptor agonists in the rat supraoptic nucleus: involvement of multiple intracellular transduction signals.

Vasopressin neurones of the supraoptic nucleus are autoregulated by vasopressin released from their soma and dendrites. Vasopressin binds to specific autoreceptors to trigger an influx of Ca(2+), and this response involves both phospholipase C (PLC) and adenylate cyclase (AC) pathways that, in the periphery, are activated by V(1) (V(1a) and V(1b))- and V(2)-type receptors. To investigate the pathways involved in the [Ca(2+)](i) response, [Ca(2+)](i) measurements were made on freshly dissociated neurones using Fura-2 microspectrofluorimetry, and vasopressin release was measured from isolated supraoptic nuclei. The [Ca(2+)](i) increase and vasopressin release induced by the V(1a) agonist were strongly inhibited by a PLC blocker, an IP(3) receptor antagonist, and a PKC blocker. An AC inhibitor did not affect the V(1a) response, while PKA inhibitors significantly reduced the V(1a)-induced [Ca(2+)](i) and release responses. The [Ca(2+)](i) increase and vasopressin release elicited by the V(2) agonist were attenuated not only by AC pathway blockers, but also by PLC inhibitors. Surprisingly, the V(1b) agonist showed no [Ca(2+)](i) or vasopressin release response. In conclusion, the V(1a) agonist activates both PLC and AC pathway, confirming the functional expression of a V(1a) vasopressin receptor on vasopressin neurones. The V(2) agonist activation of both PLC and AC pathways could result from an action on the PLC-linked unknown receptor, and/or the AC-linked dual angiotensin II-vasopressin receptor.

Impaired somatodendritic responses to pituitary adenylate cyclase-activating polypeptide (PACAP) of supraoptic neurones in PACAP type I -receptor deficient mice.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor (PAC1 receptor) in regulating hypothalamic supraoptic neurones was investigated using PAC1 receptor-deficient male mice (PAC1-/-). The effects of PACAP on [Ca2+]i were investigated in freshly dissociated supraoptic neurones and on the somatodendritic release of vasopressin and oxytocin, examined on intact supraoptic nuclei. In supraoptic neurones from wild-type mice (PAC1+/+), 100 nm PACAP induced an increase in [Ca2+]i and release of vasopressin and oxytocin, whereas in heterozygous (PAC1+/-) and null-mutant mice (PAC1-/-), PACAP was much less effective. PACAP had no effect on these two parameters when applied to isolated neurohypophysial nerve terminals of PAC1+/+ and PAC1-/- mice, and rats. In conclusion, the PAC1 receptor is solely responsible for the PACAP-induced [Ca2+]i signalling and secretion of vasopressin and oxytocin in the somatodendritic region of supraoptic neurones.

Mechanisms of cytosolic Ca2+ suppression by prostaglandin E2 receptors in rat melanotrophs.

We have previously reported that voltage-dependent Ca2+ (VDC) channels of rat melanotrophs are inhibited by prostaglandin E2 (PGE2). In this study, mechanisms involved in the inhibitory actions of PGE2 receptors of rat melanotrophs were analysed using reverse transcriptase-polymerase chain reaction (RT-PCR), Ca2+-imaging and whole-cell, patch-clamp techniques with recently developed EP agonists, each of which is selective for the known four subclasses of EP receptors (EP1-4). PGE2 reversibly suppressed the cytosolic Ca2+ concentration ([Ca2+]i). The maximum reduction in [Ca2+]i by PGE2 was comparable to that by dopamine or to that by extracellular Ca2+ removal. RT-PCR analysis of all four EP receptors revealed that EP3 and EP4 receptor mRNAs were expressed in the intermediate lobe. The effects of PGE2 to suppress [Ca2+]i were mimicked by the selective EP3 agonist, ONO-AE-248, whereas three other EP agonists, ONO-DI-004 (EP1), ONO-AE1-259 (EP2) and ONO-AE1-329 (EP4), had little or no effect on [Ca2+]i. All four G-protein activated inward rectifying K+ (GIRK) channel mRNAs were identified in intermediate lobe tissues by RT-PCR. Dopamine concentration-dependently activated GIRK currents, whereas PGE2 did not activate GIRK currents, even at the concentration causing maximal inhibition of VDC channels. These results suggest that PGE2 acts on EP3 receptors to suppress Ca2+ entry of rat melanotrophs by selectively inhibiting VDC channels of these cells. We have compared the possible cellular and molecular mechanisms of inhibition by dopamine and PGE2.

Involvement of postsynaptic EP4 and presynaptic EP3 receptors in actions of prostaglandin E2 in rat supraoptic neurones.

We have reported that supraoptic nucleus (SON) neurones are excited by prostaglandin E2 (PGE2) presumably via dual postsynaptic PG receptors, FP receptors and unidentified EP receptors, and that presynaptic EP receptors may also be involved in the excitation. In the present study, to clarify the receptor mechanism of the PGE2-mediated actions on SON neurones, we studied the pre- and postsynaptic effects of four newly developed EP agonists that are selective for each of the four EP receptors, EP1-4, on rat SON neurones using extracellular recording and whole-cell patch-clamp techniques. The EP4 agonist ONO-AE1-329 mimicked the excitatory effects of PGE2, whereas the EP1 agonist ONO-DI-004, the EP2 agonist ONO-AE1-257 and the EP3 agonist ONO-AE-248 had little or no effect. The effects of ONO-AE1-329 were unaffected by the EP1/FP/TP antagonist, ONO-NT-012, which potently suppressed the excitation caused by the FP agonist fluprostenol and PGE2. ONO-AE1-329 caused marked excitation when responses to fluprostenol were desensitized by repeated applications of fluprostenol. Patch-clamp analysis in SON neurones showed that ONO-AE1-329 induced inward currents at a holding potential of -70 mV and the reversal potential of the currents was -35.1 +/- 2.3 mV. On the other hand, the frequency of spontaneous inhibitory postsynaptic currents recorded from SON slice preparations was suppressed by ONO-AE-248, but unaffected by the other three EP agonists. These results suggest that SON neurones possess postsynaptic EP4 receptors and that gamma-aminobutyric acid neurones innervating SON neurones possess presynaptic EP3 receptors in their terminals. Activation of the two EP receptors may be involved in the excitatory regulation of SON neurones by PGE2.

Production of cAMP by adrenomedullin in human oligodendroglial cell line KG1C: comparison with calcitonin gene-related peptide and amylin.

The actions and the presence of adrenomedullin (AM) were investigated in cultured human oligodendroglial cell line KG1C. AM and AM mRNA were detected in KG1C cells by immunohistochemistry and RT-PCR. mRNAs for calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) 1, 2 and 3 but not for calcitonin receptors were detected in the cells, while mRNAs for CRLR, calcitonin receptors and all RAMPs were detected in the human cerebellum. Application of AM resulted in time- and concentration-dependent increases in the cAMP level of KG1C cells. Calcitonin gene-related peptide (CGRP) and amylin, peptides structurally related to AM, also increased cAMP. The potencies for the cAMP production of the three peptides were CGRP > or =AM >> amylin with EC(50) of 8, 18, 90 nM, respectively. The responses induced by AM were strongly inhibited by the CGRP(1) receptor antagonist human CGRP(8-37), and inhibited also by the AM receptor antagonist human AM(22-52). In contrast, the responses induced by CGRP or amylin were inhibited only by CGRP(8-37) and not by AM(22-52). The responses induced by all three peptides were unaffected by the amylin receptor antagonist human amylin(8-37). The CGRP(2) receptor agonist human [Cys(Acm)(2,7)]CGRP significantly increased the cAMP level but the increase was smaller than that caused by CGRP. This increase in cAMP was unaffected by CGRP(8-37), AM(22-52) or by amylin(8-37). These results suggest that in KG1C cells, AM increases cAMP through AM and CGRP(1) receptors, whereas CGRP does so through CGRP(1) and CGRP(2) receptors, and amylin exerts its effects through CGRP(1) receptors. Collectively, these findings imply that AM released from oligodendroglial cells may play a role in the regulation of oligodendrocytes via autocrine/paracrine through AM receptors and CGRP(1) receptors.

Elevated expression of vacuolar proton pump genes and cellular PH in cisplatin resistance.

V-ATPases are proton-translocating enzymes, which are found not only in numerous intracellular organelles but also in the plasma membranes of many eukaryotic cells. Using differential display, we have identified one of the proton pump subunit genes, ATP6C, as a cisplatin-inducible gene. Northern blot analysis demonstrated that expression of other members of the subunit is inducible by cisplatin treatment. Proton pump gene expression is also upregulated in 3 independent cisplatin-resistant cell lines but not in vincristine- or etoposide-resistant cell lines. Cellular pH was significantly higher in cisplatin-resistant cells than in sensitive parental cells. In vitro DNA-binding activity of cisplatin was markedly increased in acidic conditions, suggesting that the cytotoxicity of cisplatin is modulated by cellular pH. Furthermore, the proton pump inhibitor bafilomycin can synergistically potentiate the cytotoxicity of cisplatin but not of etoposide or camptothecin. These results indicate that cellular pH is one of the critical parameters for effective cancer chemotherapy with cisplatin.

Production of humanized antibody against human high-affinity IgE receptor in a serum-free culture of CHO cells, and purification of the Fab fragments.

We describe the preparation of Fab fragments of a humanized anti-human high-affinity IgE receptor (Fc epsilonRIalpha) antibody potentially useful for treatment of IgE-mediated allergic diseases. IgE-binding capacities of sixteen combinations of light and heavy chains of four recombinant anti-Fc epsilonRIalpha antibodies, chimeric CRA2, humanized CRA2, chimeric CRA4, and humanized CRA4, were compared. A combination in which both chains were of humanized CRA2 had the highest activity. Stable transfectant clones of four kinds of host cells expressing recombinant antibodies were established. CHO-K1 cells were the most productive. Serum-free media suitable for culture of the stable CHO-transfectant clones were screened. The concentration of the humanized CRA2, which the most productive clone secreted into the chosen serum-free medium, was approximately 100 microg/ml. A procedure for the purification of the antibody, papain-digestion, and purification of Fab fragments was established. The highly purified humanized Fab fragments are suitable for use to examine their in vivo activity and immunogenicity in primates.

Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

Design of a temporally and spatially controlled drug delivery system for the treatment of liver diseases in mice.

Strict regulation of the distribution and degradation kinetics is the ultimate aim of drug delivery system. Regulation of drug delivery would increase the therapeutic efficacy and decrease the potential side effects. We encapsulated and used Z-Asp, a caspase inhibitor in poly-N-p-vinylbenzyl-D-lactonamide (PVLA) coated-poly (L-lactic acid) (PLA)-nanospheres in a mouse model of acute hepatitis. These nanospheres were internalized and accumulated in hepatocytes both in vitro and in vivo. Encapsulation significantly extended the intracellular retention time of the content in hepatocytes, which increased the bioavailability of the caspase inhibitor. In addition, the therapeutic effect was temporally controllable in vivo by modifying the component of the nanospheres. A cocktail of nanospheres with diverse degradation kinetics showed persistent therapeutic effects in acute hepatitis, and only nanospheres that targeted hepatocytes and controlled degradation rescued mice from lethal hepatic injury. This temporally and spatially controlled drug delivery system could be used in various liver diseases.

Upregulation of synapsin IIa and IIb mRNAs in the paraventricular and supraoptic nuclei in chronic salt loaded and lactating rats.

The effects of chronic salt loading (2% saline to drink for 5 and 10 days), gestation, lactation and adrenalectomy on the expression of synapsin IIa and IIb genes were examined in the rat paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. In each control, synapsin IIa and IIb genes were moderately expressed in the magnocellular division of the PVN and SON, while few transcripts of synapsin IIa and IIb were observed in the parvocellular division of the PVN. Chronic salt loading, gestation on day 21 and lactation on day 10 caused significant increases in synapsin IIa and IIb transcripts in the magnocellular division of the PVN and SON, compared to each control. Although corticotropin-releasing hormone transcripts in the parvocellular division of the PVN were significantly increased in the adrenalectomized rats, no changes in the transcripts of synapsin IIa and IIb were observed throughout the PVN. These results suggest that physiological stimuli such as osmotic challenge and lactation potently increase synapsin IIa and IIb mRNAs in the magnocellular neurons of the PVN and SON.

Centrally administered orexin/hypocretin activates HPA axis in rats.

The effects of i.c.v. administration of orexin/hypocretin on plasma ACTH, corticosterone and c-fos mRNA in the paraventricular nucleus (PVN) of the rat were examined. Plasma ACTH levels were markedly increased at 30 min after i.c.v. administration of orexin-A. Plasma corticosterone levels were significantly increased in a dose-related manner 30 min after i.c.v. administration of orexin-A and orexin-B. In situ hybridization histochemistry revealed that the induction of the c-fos mRNA in the parvocellular division of the PVN was increased in a dose-related manner 30 min after i.c.v. administration of orexin-A and orexin-B. These results suggest that central orexin/hypocretin activates hypothalamo-pituitary-adrenal (HPA) axis and may be involved in stress-induced activation of the HPA axis.

Postnatal development of orexin/hypocretin in rats.

We examined developmental changes of orexins/hypocretins and their receptors (OX1R and OX2R) in the rat hypothalamus from postnatal day 0 to 10 weeks, using in situ hybridization histochemistry for the prepro-orexin, OX1R and OX2R mRNAs and immunohistochemistry for orexin-A and orexin-B. The prepro-orexin mRNA was weakly detected in the lateral hypothalamic area (LHA) from days 0 to 15. Orexin-A- and -B-like immunopositive cells and fibers were not detected from days 0 to 10, but they were observed after day 15. The prepro-orexin mRNA in the LHA markedly increased between days 15 and 20. The OX1R mRNA was detected in the ventromedial hypothalamic area (VMH) at day 0. The OX2R mRNA was not detected in the paraventricular nucleus (PVN) at days 0 and 1, but weakly observed on day 5. The OX1R mRNA in the VMH and OX2R mRNA in the PVN gradually increased throughout the postnatal period. Next, we examined the effects of milk deprivation and intraperitoneal (i.p.) administration of leptin on the hypothalamic prepro-orexin mRNA in pups. Although 24-h milk deprivation did not affect the level of the prepro-orexin mRNA at days 5 and 10, i.p. administration of leptin from days 0 to 3 caused a significant increase in the prepro-orexin mRNA on days 5 and 10. These results suggest that the development of orexins may be associated with developmental changes such as increase of leptin, weaning, feeding and sleep/wakefulness states.

ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells.

In human osteoblast-like MG-63 cells, extracellular ATP increased [(3)H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [(3)H]thymidine incorporation. ATP-induced [(3)H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [(3)H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced [(3)H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X(4), P2X(5), P2X(6), and P2X(7), but not P2X(1), P2X(2), and P2X(3), receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca(2+) concentration, and inhibitors for several Ca(2+)-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca(2+) concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca(2+)-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.

Induction of pituitary adenylate cyclase-activating polypeptide mRNA in the medial parvocellular part of the paraventricular nucleus of rats following kainic-acid-induced seizure.

We examined the effects of kainic acid (KA)-induced seizure on the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) gene in the paraventricular nucleus (PVN) of rats using in situ hybridization histochemistry. Subcutaneous administration of KA (12 mg/kg) in adult male Sprague-Dawley rats caused a progressive development of seizure behavior. An induction of the PACAP gene expression in the medial parvocellular part of the PVN (mpPVN) was observed 3, 6, 12, 24 and 48 h after subcutaneous administration of KA. From a nearly undetectable level, PACAP gene expression increased in the mpPVN and reached maximum 12 h after subcutaneous administration of KA. PACAP gene expression returned to near basal level 48 h after stimulation with KA. Using a specific monoclonal PACAP antibody, PACAP immunoreactivity (-IR) gradually increased during the following 24 h after KA administration. In controls, PACAP-IR was located exclusively in nerve fibers of the mpPVN, whereas KA administration induced PACAP-IR in cell bodies of the mpPVN, and a dense accumulation of PACAP-IR nerve fibers in the external zone of the median eminence was observed. Induction of the PACAP gene expression following KA-induced seizure was significantly reduced by pretreatment with diazepam or MK-801 (nonselective N-methly-D-aspartate receptor antagonist). These results suggest that PACAP in the hypothalamo-adenohypophysial system may have a hypophysiotropic role during KA-induced seizure.

Pre- and postsynaptic modulation of the electrical activity of rat supraoptic neurones.

The release of vasopressin and oxytocin is regulated by the electrical activity of magnocellular neurosecretory cells in the supraoptic and paraventricular nuclei, which is under the control of a great variety of neurotransmitters and neuromodulators. The major neural signals to the supraoptic nucleus are from excitatory glutamate inputs and inhibitory GABA inputs. In recent studies, the voltage-clamp mode of the whole-cell patch-clamp technique has been applied to slice preparations from rat hypothalamus to monitor synaptic inputs to supraoptic neurones. Spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) are abolished by CNQX and picrotoxin, respectively, but are insensitive to tetrodotoxin, indicating that they represent quantal release of glutamate and GABA, respectively, from nerve terminals of presynaptic neurones. GABA and glutamate show remarkable suppressive effects on both EPSCs and IPSCs via presynaptic GABA(B) and mGlu receptors, respectively. Noradrenaline, which excites supraoptic neurones via postsynaptic alpha1-receptors, also suppresses IPSCs and potentiates EPSCs. On the other hand, prostaglandin E2, which excites supraoptic neurones via postsynaptic prostaglandin E2 (EP) receptors of the EP4 subclass, also suppresses IPSCs via EP3 receptors but has little effect on EPSCs. Thus pre- and postsynaptic mechanisms may act cooperatively to excite supraoptic neurones. Nitric oxide, which inhibits supraoptic neurones, potentiates IPSCs without affecting EPSCs. This provides another example for the preferential modulation of IPSCs of supraoptic neurones. On the other hand, PACAP, which causes a long-lasting increase in the firing frequency via the postsynaptic receptors, has no effect on EPSCs and IPSCs, suggesting that some ligands act only at postsynaptic receptors. Thus multiple patterns for pre- and postsynaptic modulation are present in the supraoptic nucleus, and the electrical activity of supraoptic neurones is regulated via complex mechanisms at both pre- and postsynaptic sites.