Toshihiko Yanagita

Adrenomedullin regulates blood-brain barrier functions in vitro.

Adrenomedullin (AM) is an important vasodilator in cerebral circulation, and cerebral endothelial cells are a major source of AM. This in vitro study aimed to determine the AM-induced changes in blood–brain barrier (BBB) functions. AM administration increased, whereas AM antisense oligonucleotide treatment decreased transendothelial electrical resistance. AM incubation decreased BBB permeability for sodium fluorescein (mol. wt 376 Da) but not for Evans blue albumin (mol. wt 67 kDa), and it also attenuated fluid-phase endocytosis. AM treatment resulted in functional activation of P-glycoprotein efflux pump in vitro. Our results indicate that AM as an autocrine mediator plays an important role in the regulation of BBB properties of the cerebral endothelial cells.

Cerebral endothelial cells are a major source of adrenomedullin.

Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase‐polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 ± 13 fmol/105 cells per 12 h; mean ± SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte‐derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose‐dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment significantly reduced adrenomedullin production by RCECs and tended to decrease intraendothelial cAMP concentrations. These findings may suggest an important autocrine and paracrine role for adrenomedullin in the regulation of cerebral circulation and blood–brain barrier functions. Cerebral endothelial cells are a potential source of adrenomedullin in the central nervous system, where adrenomedullin can also be involved in the regulation of neuroendocrine functions.

Aquaporin subtypes in rat cerebral microvessels

We investigated the expression of aquaporin (AQP) subtypes in the rat cerebral microvessels by reverse transcription-polymerase chain reaction, immunoblotting and immunohistochemistry. mRNA for AQP4, but not for AQP1, 2, 3 or 5, was detected in the microvessels. Immunoblot analysis showed that AQP4 protein was detected as a 30 kDa band with higher molecular weight bands. Immunohistochemical staining showed that AQP4 was located on cell surface of the cerebral microvessels. These results suggest that AQP4 in the cerebral microvessels is involved in the regulation of water transport between blood and brain.

Up-regulation of sodium channel subunit mRNAs and their cell surface expression by antiepileptic valproic acid: activation of calcium channel and catecholamine secretion in adrenal chromaffin cells.

Abstract: Treatment of cultured bovine adrenal chromaffin cells with a therapeutic concentration (0.6 mM) of valproic acid (VPA) for >24 h caused a time‐dependent (t1/2 = 74 h) increase in [3H]saxitoxin binding up to 1.4‐fold without altering the KD value; it was prevented by the simultaneous treatment with cycloheximide (an inhibitor of protein synthesis). VPA also raised Na+ channel α‐ and β1‐subunit mRNA levels 1.4‐ and 1.7‐fold at 24 h, and 1.6‐ and 1.8‐fold at 72 h, respectively. Chronic (but not acute) exposure to VPA enhanced 22Na+ influx caused by various concentrations of veratridine 1.4–2.1‐fold, even when assayed in the presence of Na+,K+‐ATPase inhibitor, but did not change the EC50 value of veratridine. Ptychodiscus brevis toxin‐3 allosterically potentiated veratridine‐induced 22Na+ influx by ∼2‐fold in VPA‐treated cells as in nontreated cells. Long‐term treatment with VPA augmented veratridine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion, but had no effect on 45Ca2+ influx and catecholamine secretion caused by high K+ (a direct activation of voltage‐dependent Ca2+ channels). Chronic treatment with VPA also enhanced nicotine‐induced 22Na+ influx via the nicotinic receptor‐ion channel complex 1.2–1.4‐fold with little change in the EC50 value of nicotine, thereby increasing the nicotine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion. These results suggest that chronic treatment with VPA up‐regulates cell surface expression of Na+ channels via the transcription/translation‐dependent mechanisms, and probably of nicotinic receptors, thereby resulting in the enhancement of Ca2+ channel gating and catecholamine secretion.

Up-Regulation of Functional Voltage-Dependent Sodium Channels by Insulin in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 100 nM insulin raised [3H]saxitoxin ([3H]STX) binding in a time‐dependent manner (t1/2 = 26 h). Insulin (100 nM for 4 days) increased the Bmax of [3H]STX binding by 49% without changing the KD value and also augmented the maximal influx of 22Na+ due to 560 µM veratridine by 39% without altering the EC50 value of veratridine. The stimulatory effect of insulin on 22Na+ influx was concentration‐dependent with an EC50 of 3 nM, whereas insulin‐like growth factor (IGF)‐I had little effect at 1 nM. Ptychodiscus brevis toxin‐3 allosterically potentiated veratridine (100 µM)‐induced 22Na+ influx by approximately twofold in both insulin‐treated cells and untreated cells. Veratridine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine‐induced 22Na+ influx via the nicotinic receptor‐ion channel complex and high‐K+ (direct activation of voltage‐dependent Ca2+ channels)‐induced 45Ca2+ influx. Stimulatory effects of insulin on [3H]STX binding and veratridine‐induced 22Na+ influx were nullified by simultaneous treatment with either 5,6‐dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na+ channel α‐subunit. These results suggest that the binding of insulin to insulin (but not IGF‐I) receptors mediates the up‐regulation of functional Na+ channel expression at plasma membranes; this up‐regulation may be due, at least in part, to the de novo synthesis of an as yet unidentified protein(s).

Protein Kinase C-α and -ε Down-Regulate Cell Surface Sodium Channels via Differential Mechanisms in Adrenal Chromaffin Cells

Abstract: In cultured bovine adrenal chromaffin cells, our [3H]saxitoxin ([3H]STX) binding, immunoblot, and northern blot analyses specified protein kinase C (PKC) isoform‐specific posttranscriptional and posttranslational mechanisms that direct down‐regulation of cell surface Na channels. Immunoblot analysis showed that among 11 PKC isoforms, adrenal chromaffin cells contained only conventional (c)PKC‐α, novel (n)PKC‐ε, and atypical (a)PKC‐ζ. Treatment of adrenal chromaffin cells with 100 nM 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) or 100 nM phorbol 12,13‐dibutyrate (PDBu) caused a rapid (< 15 min) and sustained (> 15 h) translocation of PKC‐α and ‐ε (but not ‐ζ) from cytosol to membranes, whereas a biologically inactive 4α‐TPA had no effect. Thymeleatoxin (TMX), an activator of cPKC, produced similar membrane association of only PKC‐α at 100 nM, with the potency of TMX being comparable with those of TPA and PDBu. Treatment with either 100 nM TPA or 100 nM TMX reduced cell surface [3H]STX binding to a comparable extent at 3, 6, and 12 h, whereas TPA lowered the binding to a greater extent than TMX at 15, 18, and 24 h; at 15 h, Gö6976, a specific inhibitor of cPKC, completely blocked TMX‐induced decrease of [3H]STX binding while preventing by merely 57% TPA‐induced decrease of [3H]STX binding. Treatment with 100 nM TPA lowered the Na channel α‐subunit mRNA level between 3 and 12 h, with its maximum 52% fall at 6 h, and it was accompanied by a subsequent 61% rise of the β1‐subunit mRNA level at 24 h. Gö6976 failed to prevent TPA‐induced reduction of the α‐subunit mRNA level; TMX did not change the α‐and β1‐subunit mRNA levels throughout the 24‐h treatment. Brefeldin A, an inhibitor of vesicular exit from the trans‐Golgi network, augmented TPA‐ and TMX‐induced decrease of [3H]STX binding at 1 and 3 h. Our previous and present studies suggest that PKC down‐regulates cell surface Na channels without altering the allosteric gating of Na channels via PKC isoform‐specific mechanisms; cPKC‐α promotes Na channel internalization, whereas nPKC‐ε decreases the α‐subunit mRNA level by shortening the half‐life of α‐subunit mRNA without changing its gene transcription.

Adrenomedullin inhibits spontaneous and bradykinin‐induced but not oxytocin‐ or prostaglandin F2α‐induced periodic contraction of rat uterus

In isolated rat uterine strips, adrenomedullin (AM) inhibited the spontaneous periodic contraction in a concentration‐dependent manner (IC50=22.3±0.7 nM). The inhibitory effect of AM was prevented by either AM22–52, a putative antagonist for AM receptors, or calcitonin gene‐related peptide (CGRP)8–37, a putative antagonist for CGRP receptors. AM also attenuated bradykinin (BK)‐induced periodic uterine contraction, which was blocked by AM22–52 or CGRP8–37, whereas AM had no effect on the periodic contraction caused by oxytocin or prostaglandin F2α (PGF2α). RT–PCR analysis showed that mRNAs for calcitonin receptor‐like receptor (CRLR), receptor‐activity‐modifying protein (RAMP)1, RAMP2 and RAMP3 were expressed in the rat uterus. These results demonstrate that AM selectively inhibits spontaneous and BK‐induced periodic contraction via activating receptors for AM and CGRP.

Functional relation between nitric oxide and noradrenaline for the modulation of vascular tone in rat mesenteric vasculature

As previously reported, Nω-nitro-l-arginine (l-NNA), an inhibitor of nitric oxide (NO) synthesis, decreased transmural field stimulation (TFS)-induced noradrenaline overflow from the isolated perfused rat mesenteric vasculature attached to the intestine. The decrease was attenuated by l-arginine. This suggests that NO may increase noradrenaline release (Yamamoto et al. 1993).The present experiments with this preparation were done in order to monitor changes in vascular perfusion pressure caused by TFS or by noradrenaline infusion in parallel with those in the noradrenaline outflow caused by TFS in the presence of atropine (0.1 μmol/l) (to block acetylcholine-induced release of endothelial NO) and of indomethacin (3 μmol/l) (to inhibit l-NNA-induced production of vasoconstrictor prostanoids). (1) TFS (2–10 Hz) caused a frequency-dependent increase in noradrenaline overflow and perfusion pressure. (2) l-NNA (10 and 30 μmol/l) caused a concentration-dependent inhibition of TFS-induced noradrenaline overflow, whereas the TFS-induced pressure increase was augmented by l-NNA in a concentration-dependent manner. At any given concentration of l-NNA, the potentiation of vasoconstriction by l-NNA became greater in magnitude as the frequency of the TFS was raised. (3) Infusion of noradrenaline (0.38–6 nmol) caused a dose-dependent increase in perfusion pressure up to a value comparable with that caused by TITS. The pressure increase in response to noradrenaline infusion was also enhanced by l-NNA, relatively, to a greater extent than the enhancement, by l-NNA, of the pressure response to TFS. (4) These effects of l-NNA were significantly attenuated by l-arginine (0.3 mmol/l) or sodium nitroprusside (1 μmol/l). Our results suggest that NO, presumably originating from several sites, may stimulate the release of noradrenaline in the mesenteric vasculature and that the consequent rise in circulating noradrenaline, in turn, causes the liberation of endothelial NO.

REGULATION OF CELL SURFACE EXPRESSION OF VOLTAGE-DEPENDENT Nav1.7 SODIUM CHANNELS: mRNA STABILITY AND POSTTRANSCRIPTIONAL CONTROL IN ADRENAL CHROMAFFIN CELLS

Regulated expression of Na+ channels is indispensable to physiological events, whereas dysregulated expression of otherwise silent or even normal Na+ channel isoforms causes Na+ channelopathies; however, the regulatory mechanisms remain unknown. In quiescent cultured bovine adrenal chromaffin cells, constitutive phosphorylation/activation of extracellular signal-regulated kinase-1 (ERK1) and ERK2 destabilized Nav l.7 Na+ channel alpha-subunit mRNA and decreased its level without altering alpha-subunit gene transcription, thus negatively regulating steady-state level of Na+ channels. Activation of protein kinase C (PKC) down-regulated Na+ channels via PKC isoform-specific mechanisms; conventional PKC-alpha promoted endocytic internalization of Na+ channels, whereas novel PKC-epsilon destabilized alpha-subunit mRNA without altering its gene transcription. Long-lasting (but not short-term) increase of cytoplasmic Ca2+ down-regulated Na+ channels; a slowly-developing moderate increase of Ca2+ activated PKC-alpha and calpain, promoting internalization of Na+ channels, whereas an immediate monophasic and salient plateau increase of Ca2+ lowered alpha- and beta1-subunit mRNA levels. Calcineurin, or FK506 binding protein- and rapamycin-associated protein (FRAP), a serine/threonine protein kinase, down-regulated, whereas insulin receptor tyrosine kinase or protein kinase A (PKA) up-regulated, Na+ channels via modulating Na+ channel internalization, and/or Na+ channel externalization from the trans-Golgi network. Neuroprotective, antiepiletic, antipsychotic, and local anesthetic drugs up-regulated Na+ channels via transcriptional/translational events.

Adrenomedullin receptors in rat cerebral microvessels.

To characterize the sites of action of adrenomedullin (AM) in the cerebral microvasculature, we studied the effect of AM on cyclic AMP (cAMP) level as well as expression of AM and its receptor in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration technique. AM and calcitonin gene-related peptide (CGRP) increased cAMP level in the microvessels in a concentration-dependent manner. The effect of AM was more than 100 times more potent than that of CGRP. The accumulation of cAMP by AM was inhibited by AM[22-52], an AM receptor antagonist, but not by CGRP[8-37], a CGRP receptor antagonist, suggesting that AM increased cAMP accumulation by acting on receptors specific to AM. [125I]AM binding to the microvessels was displaced by AM and less potently by AM[22-52]. The displacing potencies of CGRP and CGRP[8-37] were very weak. mRNAs for AM as well as calcitonin-receptor-like receptor and receptor-activity-modifying protein 2 which form a receptor specific to AM, were highly expressed in the microvessels. These results provide biochemical and pharmacological evidence that AM is produced in and acts on the cerebral microvessels in an autocrine/paracrine manner and is involved in regulation of cerebral microcirculation.