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.

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.

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.

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.

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.

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.

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.

A physiological role for adrenomedullin in rats; a potent hypotensive peptide in the hypothalamo-neurohypophysial system.

Adrenomedullin, a potent hypotensive peptide, was originally isolated from human phaeochromocytoma. Adrenomedullin immunoreactivity and gene expression are found not only in peripheral organs but also in the central nervous system. Adrenomedullin labelled cells were localised in the hypothalamus, including in the paraventricular and supraoptic nuclei, in rats. Abundant adrenomedullin-immunoreactive fibres and varicosities were found in the hypothalamo-neurohypophysial tract and the internal zone of the median eminence in colchicine-treated and hypophysectomized rats, whereas in control rats few adrenomedullin-labelled fibres were observed. We examined the effects of intracerebroventricular administration of adrenomedullin on neurosecretory cells in the paraventricular and supraoptic nuclei of rats, using immunohistochemistry for Fos protein and in situ hybridisation histochemistry for c-fos mRNA. Intracerebroventricular administration of adrenomedullin caused a marked induction of Fos-like immunoreactivity in the paraventricular nucleus and the dorsal part of the supraoptic nucleus. In the paraventricular and supraoptic nuclei, nuclear Fos-like immunoreactivity was predominantly in oxytocin-immunoreactive cells rather than vasopressin-immunoreactive cells. The induction of c-fos mRNA in the paraventricular and supraoptic nuclei was increased in a dose-related manner 30 min after intracerebroventricular administration of adrenomedullin. This induction was reduced by pre-treatment with the adrenomedullin receptor antagonist, human adrenomedullin-(22-52)-NH2. Intracerebroventricular administration of adrenomedullin also caused a marked increase in the plasma concentration of oxytocin. Extracellular recordings from magnocellular neurosecretory cells in the paraventricular nucleus revealed that putative oxytocin-secreting cells were activated by intracerebroventricular administration of adrenomedullin. These results suggest that central adrenomedullin preferentially stimulates the secretion of oxytocin by activating hypothalamic oxytocin-secreting cells and may have an important role in salt appetite and body fluid homeostasis in rats.

Prostaglandin E2 induces Ca2+ release from ryanodine/caffeine-sensitive stores in bovine adrenal medullary cells via EP1-like receptors.

Prostaglandin E2 (PGE2) causes Ca2+ release from intracellular Ca2+ stores and stimulates phosphoinositide metabolism in bovine adrenal medullary cells. These results have been interpreted as PGE2 induces Ca2+ release from inositol trisphosphate (IP3)-sensitive stores. However, we have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), bradykinin, and angiotensin II release Ca2+ from caffeine/ryanodine-sensitive stores, although they cause a concomitant increase of intracellular IP3. In light of these results, the mechanism of PGE2-induced Ca2+ release was investigated in the present study. PGE2 dose-dependently caused a transient but consistent Ca2+ release from internal Ca2+ stores. The PGE2-induced Ca2+ release was unaffected by cinnarizine, a blocker of IP3-induced Ca2+ release. By contrast, it was potently inhibited by prior application of caffeine and ryanodine. Although IP3 production in response to PGE2 was abolished by the phospholipase C inhibitor U-73122, Ca2+ release in response to PGE2 was unaffected by U-73122. The PGE2-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A, and forskolin, a cyclic AMP (cAMP)-elevating agent, did not cause Ca2+ release. The EP1 agonist 17-phenyl-trinorPGE2 and the EP1/EP3 agonist sulprostone mimicked the Ca(2+)-releasing effects of PGE2, whereas the EP2 agonist butaprost or the EP2/EP3 agonist misoprostol caused little or no Ca2+ release. The EP1 antagonist SC-51322 significantly suppressed the Ca2+ release response induced by PGE2, whereas the EP4 antagonist AH-23828B had little effect. These results suggest that PGE2, acting on EP1-like receptors, induces Ca2+ release from ryanodine/caffeine-sensitive stores through a mechanism independent of IP3 and cAMP and that PGE2 may share the same mechanism with PACAP and the other peptide ligands in causing Ca2+ release in bovine adrenal medullary cells.

Adrenomedullin-like immunoreactivity in the rat hypothalamo-neurohypophysial tract.

Adrenomedullin-like immunoreactivity in the hypothalamo-neurohypophysial tract in colchicine-treated and hypophysectomized rats was examined by immunohistochemistry. Adrenomedullin-like immunoreactive (AM-LI) neurons were localized in the hypothalamic areas, including the paraventricular nuclei and the supraoptic nuclei. Abundant AM-LI fibers and varicosities were found in the hypothalamoneurohypophysial tract and the internal zone of the median eminence in the colchicine-treated and hypophysectomized rats, whereas in control rats few AM-LI fibers were observed. These results suggest that the axons of the AM-LI neurons in the hypothalamus may terminate in the neurohypophysis.

Acute oxidative stress modulates secretion and repetitive Ca2+ spiking in rat exocrine pancreas.

The effects of the oxidant tert-butylhydroperoxide (t-buOOH) on carbachol-stimulated pancreatic secretion in the vascularly perfused rat pancreas have been studied in parallel with [Ca2+]i signalling and amylase output in perifused rat pancreatic acinar cells. Perfusion of the pancreas with t-buOOH (0.1-1 mM) caused a rapid and irreversible inhibition of carbachol-stimulated (3x10-7 M) amylase and fluid secretion. Pre-perfusion of the pancreas with vitamin C and dithiothreitol or a cocktail of GSH and GSH-precursor amino acids provided only marginal protection against the deleterious effects of t-buOOH, even though GSH levels were elevated significantly. In perifused pancreatic acini, repetitive [Ca2+]i spikes evoked by carbachol (3x10-7 M) were sustained for 40 min. t-buOOH (1 mM) acutely increased the amplitude and duration of Ca2+ spikes, then attenuated Ca2+ spiking and subsequently caused a marked and sustained rise in [Ca2+]i. t-buOOH-induced alterations in carbachol-stimulated [Ca2+]i signalling and amylase release in perifused pancreatic acini were prevented by vitamin C. Although vitamin C restored impaired Ca2+ signalling and maintained amylase output in pancreatic acini, it seems likely that oxidative stress inhibits fluid secretion irreversibly in the intact pancreas, resulting in a loss of amylase output. Thus, perturbations in [Ca2+]i signalling may not fully explain the secretory block caused by oxidative stress in acute pancreatitis.

Centrally administered adrenomedullin increases plasma oxytocin level with induction of c-fos messenger ribonucleic acid in the paraventricular and supraoptic nuclei of the rat.

The effects of intracerebroventricular (i.c.v.) administration of adrenomedullin (AM) on plasma oxytocin (OXT), c-Fos protein (Fos), and c-fos messenger RNA (mRNA) in the paraventricular (PVN) and supraoptic nuclei (SON) of the rat were investigated using RIA for OXT, immunohistochemistry for Fos, and in situ hybridization histochemistry for c-Fos mRNA. Central administration of AM caused a significant increase in the plasma OXT level. Intracerebroventricular administration of AM caused a marked induction of Fos-like immunoreactivity (LI) in the PVN and in the dorsal parts of the SON. In the PVN and SON, OXT-LI cells predominantly exhibited nuclear Fos-LI in comparison with arginine vasopressin-LI cells. In situ hybridization histochemistry revealed that the induction of c-fos mRNA in the PVN and SON was increased in a dose-related manner 30 min after i.c.v. administration of AM. This induction was reduced by pretreatment with the AM receptor antagonist, human AM-(22-52)-NH2. These results suggest that central AM is responsible for activating the neurosecretory cells in the PVN and SON via selective AM receptors, and that AM stimulates the secretion of OXT by activating hypothalamic OXT-producing cells.

Effects of centrally administered pituitary adenylate cyclase-activating polypeptide on c-fos gene expression and heteronuclear RNA for vasopressin in rat paraventricular and supraoptic nuclei.

The effects of intracerebroventricular (i.c.v.) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) on the expression of c-fos gene as well as heteronuclear (hn) RNA for arginine vasopressin (AVP) in paraventricular (PVN) and supraoptic nuclei (SON) of rats were investigated by immunohistochemistry for c-fos protein (Fos) and in situ hybridization histochemistry for c-fos mRNA and AVP hnRNA. The i.c.v. administration of PACAP (200 pmol/rat) caused a marked induction of Fos-like immunoreactivity (LI) in PVN and SON. The nuclear Fos-LI existed in AVP-LI containing cells in the PVN and SON. The expression of the c-fos gene in the PVN and SON was increased in a dose-related manner 30 min after i.c. v. administration of PACAP. PACAP-induced expression of the c-fos gene was significantly reduced by pretreatment with a PACAP receptor antagonist, PACAP-(6-38)-NH2. In addition, Fos-LI and the expression of the c-fos gene were also observed in the periventricular region of the third ventricle after i.c.v. administration of PACAP. The induction of c-fos gene expression in the PVN and SON reached a maximum 30 min after PACAP administration. The expression of c-fos gene in the PVN and SON induced by i.c.v. administration of vasoactive intestinal peptide (200 pmol/rat) was weaker than that induced by PACAP. The hnRNA for AVP in the PVN and SON was significantly increased 30 min after i.c.v. administration of PACAP (200 pmol/rat). Our results suggest that PACAP activates PVN and SON neurons via PACAP receptors and, in parallel, transcription of the AVP gene in the PVN and SON.

Evidence that multiple P2X purinoceptors are functionally expressed in rat supraoptic neurones.

1. The expression, distribution and function of P2X purinoceptors in the supraoptic nucleus (SON) were investigated by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and Ca2+-imaging and whole-cell patch-clamp techniques, respectively. 2. RT-PCR analysis of all seven known P2X receptor mRNAs in circular punches of the SON revealed that mRNAs for P2X2, P2X3, P2X4, P2X6 and P2X7 receptors were expressed in the SON, and mRNAs for P2X3, P2X4 and P2X7 were predominant. 3. In situ hybridization histochemistry for P2X3 and P2X4 receptor mRNAs showed that both mRNAs were expressed throughout the SON and in the paraventricular nucleus (PVN). 4. ATP caused an increase in [Ca2+]i in a dose-dependent manner with an ED50 of 1.7 x 10-5 M. The effects of ATP were mimicked by ATPgammaS and 2-methylthio ATP (2MeSATP), but not by AMP, adenosine, UTP or UDP. alphabeta-Methylene ATP (alphabetaMeATP) and ADP caused a small increase in [Ca2+]i in a subset of SON neurones. 5. The P2X7 agonist 2'- & 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) at 10-4 M increased [Ca2+]i, but the potency of BzATP was lower than that of ATP. In contrast, BzATP caused a more prominent [Ca2+]i increase than ATP in non-neuronal cells in the SON. 6. The effects of ATP were abolished by extracellular Ca2+ removal or by the P2 antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and inhibited by extracellular Na+ replacement or another P2 antagonist, suramin, but were unaffected by the P2X7 antagonist oxidized ATP, and the inhibitor of Ca2+-ATPase in intracellular Ca2+ stores cyclopiazonic acid. 7. Two patterns of desensitization were observed in the [Ca2+]i response to repeated applications of ATP: some neurones showed little or moderate desensitization, while others showed strong desensitization. 8. Whole-cell patch-clamp analysis showed that ATP induced cationic currents with marked inward rectification. The ATP-induced currents exhibited two patterns of desensitization similar to those observed in the [Ca2+]i response. 9. The results suggest that multiple P2X receptors, including P2X3, are functionally expressed in SON neurones, and that activation of these receptors induces cationic currents and Ca2+ entry. Such ionic and Ca2+-signalling mechanisms triggered by ATP may play an important role in the regulation of SON neurosecretory cells.

Neuronal nitric oxide synthase gene expression in human pituitary tumours: a possible association with somatotroph adenomas and growth hormone-releasing hormone gene expression.

OBJECTIVE: Nitric oxide (NO) has been implicated in the control of the secretory response to growth hormone-releasing hormone (GHRH) and may also modify GH release in response to excitatory aminoacids. Although rat and mouse pituitary cell lines have been shown to express neuronal NO synthase (nNOS), there has until now been no information on nNOS gene expression in human pituitary adenomas. Our objective was to provide such data and correlate the presence of nNOS transcripts with GHRH transcripts. PATIENTS: Pituitary adenoma tissue was obtained from a random selection of 32 patients with somatotrophadenomas, 16 patients with corticotroph adenomas, 39 patients with endocrinologically inactive adenomas and nine patients with macroprolactinomas undergoing transsphenoidal hypophysectomy. MEASUREMENTS: Transcripts for nNOS and GHRH were identified in frozen tissue sections by in situ hybridization histochemistry using synthetic 35S-labelled oligodeoxynucleotide probes with 100% homology to the target transcript. RESULTS: Neuronal NOS transcripts were identified in one of 16 corticotroph adenomas (6%), one of nine macroprolactinomas (11%), six of 39 endocrinologically inactive adenomas (15%) and 13 of 32 somatotroph adenomas (41%). GHRH transcripts were found in a similar distribution to nNOS transcripts in 10 of the 13 nNOS-expressing somatotroph adenomas, and in three of the four remaining adenomas from which suitable tissue was available. Cross-hybridization of the nNOS and GHRH probes to the same target was excluded by including rat brain sections cut through the arcuate nucleus as hybridization controls. Furthermore, two different nNOS oligodeoxynucleotide probes complementary to different regions of the target transcript produced identical results. CONCLUSIONS: These results suggest that there is a close correlation between nNOS gene expression and 'ectopic' expression of GHRH in human pituitary tumours, especially somatotroph adenomas. The relevance of these findings from a functional or pathologenic viewpoint remains unclear, but the data again emphasize that it is not just GH secretion that distinguishes somatotroph adenomas from other pituitary tumours.

Patch-clamp analysis of the mechanism of PACAP-induced excitation in rat supraoptic neurones.

In neurosecretory cells of the supraoptic nucleus (SON) of rats, pituitary adenylate cyclase activating polypeptide (PACAP) causes an increase in [Ca2+]i, and stimulates somatodendritic vasopressin (VP) release. In this report, to elucidate the ionic mechanism of the action of PACAP, membrane potentials and ionic currents were measured from SON neurones in slice preparations or from dissociated SON neurones. In the current clamp mode, PACAP depolarized membrane potentials of both phasic and non-phasic neurones and increased the firing rate. Moreover, simultaneous measurements of membrane potentials and [Ca2+]i revealed that the membrane depolarization correlated well with increases in [Ca2+]i. In the voltage-clamp mode, PACAP induced inward currents at a holding potential of -70 or -80 mV in a dose-dependent manner and the time course of the currents was similar to that of the PACAP-induced membrane depolarization. The averaged reversal potential of the PACAP-induced currents obtained from dissociated SON neurones was -33 mV, which was close to the reversal potential of non-selective cation currents in SON neurones. The currents were rapidly and reversibly inhibited by a cation-channel blocker, gadolinium. Analysis of synaptic inputs into SON neurones in slice preparations revealed that PACAP had little or no effects on the frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results suggest that pituitary adenylate cyclase activating polypeptide (PACAP) activates PACAP receptors in the postsynaptic membrane of the supraoptic nucleus (SON) neurones, and that the activation of PACAP receptors leads to opening of non-selective cation channels, depolarization of the membrane potential, and increase in the firing rate in SON neurones. Such mechanisms may account for the PACAP-induced increase in [Ca2+]i and vasopressin (VP) release observed in SON neurones.