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Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca2+ concentration ([Ca2+]i) from single dissociated SON neurons. PACAP at concentrations from 10(-12) to 10(-7) M increased [Ca2+]i in dissociated SON neurons in a dose-dependent manner. The patterns of the PACAP-induced [Ca2+]i increase were either sustained increase or cytosolic Ca2+ oscillations. PACAP (10[-7] M) increased [Ca2+]i in 27 of 27 neurons and glutamate (10[-4] M) increased [Ca2+]i in 19 of 19 SON neurons examined, whereas angiotensin II (10[-7] M) increased [Ca2+]i in only 15 of 60 SON neurons examined. PACAP at lower concentrations (10[-10] to 10[-8] M) increased [Ca2+]i in 70-80% of neurons examined. Although the onset and recovery of the PACAP-induced [Ca2+]i increase were slower than those observed with glutamate, the spatial distribution of the [Ca2+]i increases in response to the two ligands were similar: [Ca2+]i increase at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP-induced [Ca2+]i responses were abolished by extracellular Ca2+ removal, the L-type Ca2+-channel blocker, nicardipine, or by replacement of extracellular Na+ with N-methyl D-glucamine, and were partially inhibited by the Na+-channel blocker, tetrodotoxin. The N-type Ca2+-channel blocker, omega-conotoxin GVIA did not significantly inhibit the PACAP-induced [Ca2+]i responses. Furthermore, PACAP (10[-7] M) as well as glutamate (10[-4] M) increased AVP release from SON slice preparations, and extracellular Ca2+ removal or nicardipine inhibited the AVP release in response to PACAP. These results indicate that PACAP enhances Ca2+ entry via voltage-gated Ca2+ channels and increases [Ca2+]i, which, in turn, stimulates somatodendritic vasopressin release by directly activating PACAP receptors on SON neurons. The results also suggest that PACAP in the SON may play a pivotal role in the control of the neurohypophyseal function at the level of the soma or the dendrites.

Background and purpose: Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-go-go-related gene (hERG) K+ channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein. Experimental approach: Wild-type hERG and SCN5A/hNav 1.5 Na+ channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K+ and Na+ currents were recorded in these cells by using the whole-cell patch-clamp technique (23°C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry. Key results: Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNav 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking. Conclusions and implications: Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs. British Journal of Pharmacology (2008) 153, 439–447; doi:10.1038/sj.bjp.0707537; published online 29 October 2007

Prostaglandin E2 (PGE2) has been implicated in the excitatory regulation of magnocellular neurones in the supraoptic nucleus (SON). We have recently reported that PGE2 excited SON neurones by directly activating postsynaptic PGE2 receptors (EP receptors) of a subclass other than EP1-3, but did not affect excitatory postsynaptic currents (EPSCs). In the present study, we examined presynaptic effects of PGE2 on rat SON neurones by measuring spontaneous inhibitory postsynaptic currents (IPSCs) by a slice patch-clamp technique. PGE2 inhibited spontaneous IPSCs in a dose-dependent and reversible manner. PGE2 selectively suppressed the frequency of IPSCs without affecting the amplitude of IPSCs in the presence of tetrodotoxin, a blocker of Na+ channels, indicating that the effects were presynaptic. The inhibitory effects of PGE2 on the frequency of IPSCs were mimicked by the EP1/EP3 agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, whereas the EP2 agonist, butaprost, or the FP agonist, fluprostenol, had little effect. The effects of PGE2 on IPSCs were unaffected by the selective EP1 antagonist, SC-51322. They were unaffected also by antagonists of GABAB and alpha2 adrenergic receptors, which are present at presynaptic terminals of GABA neurones in the SON and cause suppression of spontaneous IPSCs. The inhibitor of PG synthesis, indomethacin, had little effect on spontaneous IPSCs and on the inhibitory effects of PGE2 as well as of the GABAB agonist, baclofen, and noradrenaline. These results suggest that PGE2 inhibits release of GABA from the GABAergic terminals innervating SON neurones by activating presynaptic EP receptors, presumably of the EP3 subclass, and that such a presynaptic mechanism may play a role in the excitatory regulation of SON neurones by PGE2.

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.

Mizoribine (MZR) has shown to be effective against antineutrophil cytoplasmic antibody (ANCA)-related vasculitis; however, no reports have described the successful treatment of steroid-resistant ANCA-related vasculitis with MZR in patients with renal insufficiency requiring hemodialysis. We herein report the case of a 39-year-old man undergoing hemodialysis in whom MZR successfully lowered the myeloperoxidase (MPO)-ANCA titer accompanied by remission of interstitial pneumonia, together with the pharmacokinetics of MZR. The patient developed severe renal insufficiency and interstitial pneumonia, and was started on hemodialysis. Although prednisolone was administered followed by azathioprine, the MPO-ANCA level and interstitial pneumonia showed insufficient improvement. Azathioprine was replaced by MZR and the administered dose of MZR was determined by measuring serum concentrations of MZR at the start of the dialysis session; this was because we confirmed that MZR could only be removed via dialysis, and that the serum concentration of MZR was maintained until the next dialysis session. The maintenance dose was finally set at MZR 75 mg after each dialysis. Subsequently, the ANCA titer decreased and interstitial pneumonia resolved without any MZR-related side effects. This case demonstrates that MZR is safe and effective, even in patients with steroid-resistant ANCA-related vasculitis undergoing hemodialysis, and can be monitored by measuring serum concentrations of MZR.