Teruo Nakadate

α2-Adrenoceptors modulating insulin release from isolated pancreatic islets

SummaryUsing rat isolated pancreatic islets, we investigated the effects of various α-adrenoceptor blocking agents on adrenaline-induced inhibition of glucose-stimulated insulin release. Yohimbine was about 100 times more potent than prazosin in antagonizing the inhibitory effect of adrenaline. At concentrations of 10 μM, phentolamine was about as effective as an antagonist as yohimbine, whereas dihydroergotamine, WB-4101 and phenoxybenzamine were less effective and prazosin produced very little antagonism. These results strongly suggest that postsynaptic α2-adrenoceptors modulate insulin release from pancreatic islets.

Regulation of plasma insulin level by α2-adrenergic receptors

Phentolamine, yohimbine or dihydroergotamine markedly increased plasma immunoreactive insulin (IRI) and inhibited epinephrine-induced hyperglycemia in fasted mice. On the other hand, phenoxybenzamine or prazonsin only slightly increased plasma IRI and enhanced epinephrine-induced hyperglycemia. These results indicate that there is a distinct difference in the effects of α-adrenergic blockers on the plasma IRI and glucose levels, and that α-adrenergic receptors responsible for the plasma IRI level resemble α2-adrenergic receptors more closely.

Prevention of glucose-induced insulin secretion by lipoxygenase inhibitor

Glucose-induced insulin secretion was investigated using isolated pancreatic islets of rats. The phospholipase A2 inhibitors. p-bromophenacyl bromide (0.1 mM) and mepacrine (0.1 mM) inhibited glucose-induced insulin secretion. Indomethacin (5 microM), a cyclooxygenase inhibitor, failed to inhibit glucose-induced insulin secretion, while the lipoxygenase inhibitor nordihydroguaiaretic acid (0.1-0.2 mM) inhibited it. These results suggest that stimulation of phospholipase A2 and a product(s) formed by the lipoxygenase pathway play an important role in the glucose-induced insulin secretion.

?2-Adrenoceptors inhibit the cholera-toxin-induced intestinal fluid accumulation

SummaryThe effects of adrenoceptor agonists and antagonists on the cholera-toxin-induced intestinal fluid accumulation and the mucosal levels of cAMP were investigated in vivo. Cholera toxin produced a marked fluid accumulation. Adrenaline inhibited the effect of the toxin in a dose-dependent manner. An α1-adrenoceptor blocking agent yohimbine antagonized the effect of adrenaline. The α1-adrenoceptor blocking agents prazosin and phenoxybenzamine failed to antagonize the effect of adrenaline. A high dose of a β-adrenoceptor blocking agent pindolol did not antagonize the effect of adrenaline. Yohimbine or pindolol alone did not produce any effects on the toxin-induced fluid accumulation. However, prazosin and phenoxybenzamine per se inhibited the toxin-induced fluid accumulation. An α2-selective agonist clonidine was slightly more potent than adrenaline, and was about 100-fold more potent than the α1-selective agonist methoxamine in inhibiting the cholera-toxin-induced intestinal secretion. Clonidine, adrenaline and methoxamine failed to reduce the mucosal levels of cAMP, while these α-adrenoceptor agonists inhibited the toxin-induced fluid accumulation in the same preparations. These results suggest that the stimulation of α2-adrenoceptors inhibit the cholera-toxin-induced intestinal secretion without reducing the whole mucosal levels of cAMP.

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced insulin secretion: Inhibition by phospholipase A2- and lipoxygenase-inhibitors

Abstract In isolated pancreatic islets of rats, an insulin secretion was induced by a tumor promoter 12-o-tetradecanoylphorbol-13-acetate in a low glucose medium. The insulin secretion was inhibited by p-bromophenacyl bromide, mepacrine, nordihydroguaiaretic acid and 1-phenyl-3-pyrazolidinone but not by indomethacin. When the insulin secretion was suppressed by p-bromophenacyl bromide, the secretion was partially but not significantly restored by lysophosphatidyl choline and was fully restored by the simultaneous addition of arachidonic acid and lysophosphatidyl choline. These results suggest that activation of phospholipase A 2 and a lipoxygenase product(s) play important roles in the 12-o-tetradecanoylphorbol-13-acetate-induced insulin secretion.

Inhibition of mouse epidermal 12‐lipoxygenase by 2,3,4‐trimethyl‐6‐(12‐hydroxy‐5,10‐dodecadiynyl)‐l,4‐benzoquinone (AA861)

2,3,5‐Trimethyl‐6‐(12‐hydroxy‐5, 10‐dodecadiynyl)‐1.4‐benzoquinone (AA861) strongly inhibited epidermal lipoxygenase activity which was determined by the formation of [14 C]12‐hydroxy‐5,8,10,14‐eicosatetraenoic acid by incubating [l4 C]arachidonic acid with cytosol fraction of epidermal homogenate of ***CD‐1 mice. AA861 failed to inhibit epidermal cyclooxygenase activity. The present results indicate that AA861 is a potent inhibitor of 12‐lipoxygenase in mouse epidermis (IC50 1.9 μM).

Inhibition of dibutyryl cyclic amp-induced insulin release by alpha-2 adrenergic stimulation

Effects of alpha adrenergic agonists and antagonists on dibutyryl cAMP (Bt2cAMP)-induced insulin release were investigated with isolated rat pancreatic islets. Bt2cAMP (4 mM) produced 2-fold stimulation of insulin release in all the concentrations of glucose examined (3.3-16.7 mM). Clonidine but not phenylephrine inhibited the Bt2cAMP-stimulated insulin release in a concentration-dependent manner with approximate EC50 of nanomolar range. Yohimbine but not prazosin antagonized the inhibitory effect of clonidine on the Bt2-cAMP-induced insulin release. These results suggest that alpha-2 adrenergic mechanisms are involved in a step distal to cAMP generation.

Insulinotropic effects of exogenous phospholipase A2 and C in isolated pancreatic islets

In isolated pancreatic islets of rats, exogenous phospholipase A2 produced concentration-dependent insulin secretion. p-Bromophenacyl bromide-treated enzyme no longer showed the insulinotropic effect. The insulinotropic effect of phospholipase A2 was inhibited by nordihydroguaiaretic acid (NDGA) and 1-phenyl-3-pyrazolidinone (Phenidone) but not by indomethacin. Exogenous phospholipase C also demonstrated a concentration-related insulinotropic effect. The phospholipase C-induced insulin secretion, however, was inhibited by none of the above inhibitors. These results indicate that a lipoxygenase product(s) is involved in the insulinotropic action of phospholipase A2.

Differential effects of various skin tumor-promoting agents on prostaglandin E2 release from primary cultures of mouse epidermal cells

Prostaglandin E2 (PGE2) release from primary cultures of mouse epidermal cells was markedly stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein and 1-oleoyl-2-acetyl-glycerol but not by 4 alpha-phorbol-12,13-di-decanoate in low Ca2+ (50 microM) medium. TPA-evoked PGE2 release was inhibited by mepacrine, indomethacin and H-7 but not by HA1004. These findings suggest that TPA stimulates PGE2 release through activation of protein kinase C, phospholipase A2 and the cyclooxygenase pathway. Of the non-TPA type of tumor promoting agents, i.e. anthralin, chrysarobin, 7-bromomethylbenz[a]anthracene, benzoylperoxide, okadaic acid and palytoxin, only anthralin stimulated PGE2 release. Anthralin-evoked PGE2 release was not inhibited by H-7. In normal Ca2+ (1.8 mM) medium, PGE2 release increased markedly compared to the release in low Ca2+ medium. In normal Ca2+ medium, PGE2 release was stimulated by TPA, anthralin and okadaic acid but not by other tumor promoting agents. In mouse peritoneal macrophages, TPA, palytoxin and okadaic acid stimulated PGE2 release, but other tumor-promoting agents failed to stimulate it. These results suggest that skin tumor promoting agents are not necessarily effective stimulators of prostaglandin production either in macrophages or in epidermal cells, the target cells of skin tumor promotion.

Some properties of lipoxygenase activities in cytosol and microsomal fractions of mouse epidermal homogenate

Lipoxygenase activity in microsomal fraction of mouse epidermal homogenate was characterized in comparison with cytosol lipoxygenase activity. The major activity was identified as 12-lipoxygenase in microsomal fraction as well as in cytosol fraction by the analyses with high-performance liquid chromatography and gas chromatography-mass spectrometry. Apparent Km values of cytosol and microsomal 12-lipoxygenase for arachidonic acid were 5.0 microM and 6.2 microM respectively. Apparent Vmax values were 14 pmol/min/mg protein for the cytosol enzyme and 32 pmol/min/mg protein for the microsomal enzyme. Net activities of cytosol and microsomal 12-lipoxygenase were 214 and 109 pmol/min/g wet tissue, respectively. Both cytosol and microsomal lipoxygenase activities were neither dependent on calcium nor ATP. Carbon monoxide failed to affect these enzyme activities. There were considerable differences either in the effect of glutathione or in the sensitivities toward several lipoxygenase inhibitors, indicating that the cytosol and the microsomal 12-lipoxygenase activities are derived from two different enzymes. Alternatively, the differences could be attributable to the different microenvironments of these enzymes.