Yoshikazu Nakazato

Permeability characteristics of hemoglobin derivatives across cultured endothelial cell monolayers

To better understand the vascular activity of hemoglobin-based (Hb-based) oxygen carriers, the endothelial permeability characteristics of Hb derivatives having various molecular masses were defined by using monolayers of bovine endothelial cells cultured on microporous membranes. The endothelial permeability of unmodified bovine Hb was almost twice that of bovine serum albumin. Intramolecularly cross-linked human Hb showed slightly but significantly reduced permeability as compared with unmodified bovine Hb. Polyethyleneglycol modification or haptoglobin binding to Hb further reduced the permeability. These properties were intensified in conditions in which the endothelial barrier function was reduced by pretreatment with either interleukin-6 (100 ng/mL, 21 hours) or lipopolysaccharide (1 microg/mL, 10 hours). In contrast, there was little permeability of liposome-encapsulated Hb, and it was almost unaffected by the pretreatments. These data provide the first information that Hb derivatives with smaller molecular masses show larger transendothelial flux. Because Hb is a potent scavenger of endothelium-derived relaxing factor (EDRF), our observations support the idea that smaller Hb-based acellular oxygen carriers are potent vasoconstrictors as a result of abluminal EDRF scavenging.

The mode of action of caffeine on catecholamine release from perfused adrenal glands of cat

1 Adrenaline and noradrenaline secretion induced by caffeine was investigated in the perfused cat adrenal glands. 2 Caffeine (10–80 mm) caused a dose‐dependent increase in both adrenaline and noradrenaline secretion when applied for 1 min and 10 min after replacing Ca2+ with 10−5m EGTA in the perfusion solution. The ratio of adrenaline to noradrenaline was about 1:1. Mg2+ and/or Ca2+ inhibited the response to caffeine. 3 When caffeine (40 mm) was repeatedly applied in the absence of extracellular Ca2+, the secretory response almost disappeared but only at the second challenge with caffeine. However, the response was partially restored after readmission of Ca2+ (2.2 mm) and was augmented after the readmission of Ca2+ with ouabain (10−5m). 4 Caffeine‐induced secretion of adrenaline and noradrenaline increased with the increase in the preloaded concentration of Ca2+ and attained a maximum at 16 mM Ca2+. 5 During perfusion with Ca2+‐free Locke solution containing hexamethonium (10−3m), acetylcholine (10−4m) caused increases in both adrenaline and noradrenaline secretions with a ratio of about 1:2. The secretory responses were partially inhibited by preceding stimulation with exposure to caffeine (80 mm). 6 These results suggest that caffeine mobilizes Ca2+ from an intracellular storage site that may not be entirely the same as that linked to muscarinic receptors, and causes an increase in both adrenaline and noradrenaline secretion from cat adrenal chromaffin cells.

Ryanodine inhibits caffeine-evoked Ca2+ mobilization and catecholamine secretion from cultured bovine adrenal chromaffin cells.

The effects of ryanodine, a selective inhibitor of the Ca2+‐induced Ca2+ release mechanism, on caffeine‐evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and cate‐cholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5–40 mM) caused a concentration‐dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg2+‐free medium containing 0.2 mM EGTA. Ryanodine (5 × 10–5M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 × 10–7–5 × 10–5M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2/Mg2+‐free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin‐permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.

Effects of Monensin and Veratridine on Acetylcholine Release and Cytosolic Free Ca2+ Levels in Cerebrocortical Synaptosomes of Rats

Monensin (10−8‐10−4M) caused a dose‐dependent increase in the release of [3H] acetylcholine ([3H] ACh) from purified rat cerebrocortical synaptosomes, with an EC50 of ∼1.6 × 10−6M. Extracellular Na+, but not Ca2+, was required for a monensin‐induced increase in the release of [3H] ACh. Monensin also increased the cytosolic free Ca2+ concentration ([Ca2+]i) and uptake of 22Na+ in a dose‐dependent manner. Monensin continued to cause a dose‐dependent increase in [Ca2+]i in the absence of extracellular Ca2+, although an ∼50% reduction was noted at concentrations of >10−5M. The EC50 for the monensin‐induced increase in [Ca2+]i was similar to that noted in the release of [3H]ACh. Veratridine exhibited effects similar to those of monensin, but a large portion of the increase in [Ca2+]i and [3H] ACh release was dependent on extracellular Ca2+. Measurements of rhodamine 6G fluorescence indicated that monensin and veratridine caused synaptosomal hyperpolarization and depolarization, respectively. Tetrodotoxin (10−6M) completely blocked all the effects of veratridine but had no effect on the activity of monensin. These results suggest that monensin increases the release of ACh at least in part by increasing [Ca2+]i, resulting from the increase in the Na+ influx through tetrodotoxin‐insensitive mechanisms in rat cerebrocortical synaptosomes.

Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; KT−4‐10−6 M) caused a dose‐dependent increase in the release of [3H] acetylcholine ([3H] ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose‐response curves for the PTX‐induced increases in [3H] ACh release and in [Ca2+]iwere depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H] ACh induced by concentrations of PTX < 10−10M was more dependent on the simultaneous presence of both Ca2+ and Na+than the release induced by higher concentrations of PTX. The PTX‐induced increase both in [3H] ACh release and in {Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10−6M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin‐insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H] ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na+‐Ca2+ exchange mechanism.

The role of Na+ in muscarinic receptor-mediated catecholamine secretion in the absence of extracellular Ca2+ in cat perfused adrenal glands

1 The role of Na+ in muscarinic receptor‐mediated catecholamine secretion, which is independent of extracellular Ca2+, was investigated by observing the effect of veratridine and ouabain in perfused adrenal glands of the cat. 2 Veratridine (10−4m) markedly enhanced catecholamine secretion evoked by acetylcholine (ACh, 10−4m) during perfusion with Ca2+‐free Locke solution containing hexamethonium (10−3m). The enhancement tended to be larger for noradrenaline secretion than for adrenaline secretion. Qualitatively the same result was obtained in the response to pilocarpine (5 × 10−4m). 3 Ouabain (10−4m) also enhanced ACh‐ and pilocarpine‐induced catecholamine secretions, especially noradrenaline secretion in the absence of extracellular Ca2+. 4 Tetrodotoxin (10−6m) blocked the enhancing effect of veratridine on ACh‐induced catecholamine secretion, but not that of ouabain in the absence of extracellular Ca2+. 5 When NaCl was replaced with sucrose, there was no secretory response to ACh regardless of the presence or absence of veratridine or ouabain. However, when ouabain, but not veratridine, was infused with Na+ before the replacement of NaCl, the response to ACh was substantially augmented. 6 These results indicate that Na+ is essential in the initiation of muscarinic receptor‐mediated catecholamine secretion and its enhancement by veratridine and ouabain in the absence of extracellular Ca2+. Both drugs seem to increase the intracellular concentration of Na+ through different mechanisms and result in increases in the efficiency of Ca2+ mobilization from intracellular Ca2+ pools linked to muscarinic receptors.

[3H]acetylcholine release and the change in cytosolic free calcium level induced by high K+ and ouabain in rat brain synaptosomes ☆

High K+ (50 mM) increased both [3H]acetylcholine ([3H]ACh) release and cytosolic free calcium level ([Ca2+]i) in rat brain synaptosomes in the presence of extracellular Ca2+. Ouabain (5 x 10(-8) to 5 x 10(-4) M) also caused a dose-dependent increase in [3H]ACh release, but not in [Ca2+]i, in the absence of Ca2+. The effects of high K+ and ouabain on [3H]ACh and/or [Ca2+]i, were inhibited by the intracellular Ca2+ antagonist TMB-8 (10(-4) M). These results suggest that unlike high K+, ouabain increases transmitter release from nerve endings through a mechanism which is independent of [Ca2+]i, but sensitive to TMB-8.

Sodium ions inhibit the stimulant action of caffeine on catecholamine secretion from adrenal chromaffin cells of the guinea pig

Catecholamine secretion evoked by caffeine (40 mM) was markedly enhanced by replacing NaCl in the medium with sucrose or KCl in the absence, but not in the presence, of extracellular Ca2+ and Mg2+ in both perfused adrenal glands and isolated chromaffin cells of the guinea pig. The response to caffeine declined on repetition, but was restored completely after readmission of Ca2+. These results indicate that extracellular Na+ inhibits caffeine from stimulating catecholamine secretion, which may be mediated by a release of Ca2+ from intracellular storage sites in the adrenal chromaffin cells in the presence of extracellular Ca2+ and/or Mg2+.

Relaxant mechanisms of parathyroid hormone in rat mesenteric artery.

The effects of parathyroid hormone (PTH) on tension and intracellular Ca2+ level ([Ca2+]in) were examined in ring preparations of rat mesenteric artery using isometric tension recording and the fura-2 method, respectively. The PTH (30 n M) elicited relaxation in arterial rings precontracted by phenylephrine regardless of the presence or absence of endothelium. In the endothelium-denuded arterial rings precontracted by 3 &mgr;M of phenylephrine or 60 m M of potassium chloride (KCl), PTH-related protein and PTH produced concentration-dependent relaxation to the same extent, but inhibited contraction induced by phenylephrine more effectively than that induced by KCl. Phenylephrine-induced tonic contraction was changed to a phasic one with decreased peak tension in the presence of PTH. Similar changes were observed with extracellular Ca2+ removal or methoxyverapamil plus SK&F96365, respective of voltage-gated and receptor-operated Ca2+ channel inhibitors. Phenylephrine evoked a concentration-dependent contraction concomitant with an increase in [Ca2+]in. PTH reduced both responses to the same extent. In a Ca2+-free solution, PTH inhibited a phasic contraction and a transient increase in [Ca2+]in in response to phenylephrine but not caffeine. Reverse transcriptase–polymerase chain reaction showed that PTH1 and PTH2 receptors were expressed in the rat mesenteric artery. In this tissue, PTH increased cyclic adenosine monophosphate (cAMP) levels. These results suggest that the inhibitory effect of PTH on &agr;1-adrenoceptor-mediated contraction results from the inhibition of Ca2+ influx through receptor-operated and voltage-gated Ca2+ channels, and Ca2+ release from Ca2+ stores, probably via increased cAMP in the rat mesenteric artery.