Impaired NO release from bovine aortic endothelial cells exposed to activated platelets.

We have previously shown that aggregated human platelets elicited a decrease in intracellular adenosine triphosphate (ATP), enhanced adenosine egress and damage to mitochondria in bovine aortic endothelial cells (ECs). To test whether such metabolic and ultrastructural changes could be associated with functional impairment of ECs, we investigated the effects of activated platelets on nitric oxide (NO) and prostacyclin release, and on the antiaggregation property of ECs. Pretreatment of ECs with aggregated platelets transiently stimulated basal NO release while prolonged (> or = 30 min) exposure dose-dependently inhibited NO release, both basal and in response to ATP or serotonin, with NO synthase activity being attenuated in these cells. Supplementary L-arginine (L-A) restored NO release completely. Prostacyclin release was also stimulated transiently but not affected by prolonged pretreatment. The antiaggregation property of ECs was attenuated by pretreatment with activated platelets but restored with L-A supplement. Although the effects of activated platelets and 0.5 mM acetylsalicylic acid (ASA) to attenuate the antiaggregation property of ECs were additive, activated platelets had no effect on ECs treated with 0.2 mM N omega-nitro-L-arginine (L-NA), suggesting a common mechanism. We conclude that prolonged exposure to aggregated platelets may affect the antiaggregation property of ECs by directly inhibiting NO synthesis, which may be normalized by L-A supplementation.

Three-dimensional microvasculature of the hair follicle.

The three-dimensional microvasculature of the hair follicle of the adult Wistar rat was demonstrated by scanning electron microscopy of vascular corrosion casts. The anagen hair follicle was surrounded by the basket-like capillary network which was supplied by the branches of the subcutaneous artery and drained into the veins continuous with the subcutaneous vein. The capillary network surrounding the anagen hair follicle was most dense at its bottom, and became sparse at its upper part. The telogen hair follicle was surrounded by only a few capillaries. Transmission electron microscopy showed that the capillaries around the hair bulb possessed fenestrations. Our results indicate that the microvasculature of the anagen hair follicle is so organised as to supply the hair bulb with abundant blood, which is the most important area for hair growth.

Acute vigorous exercise attenuates sensitivity of platelets to nitric oxide.

We tested whether inhibition of platelet activation by nitric oxide (NO) might be altered by strenuous exercise. Sixteen healthy male non-smokers, aged 20-26 years, underwent treadmill testing. All subjects reached Bruce stage IV without chest pain or abnormal ST-T wave changes. Platelet aggregation by Born's method and cyclic GMP accumulation in the washed platelets were determined before and immediately after exercise. Dose-response curves for platelet aggregation by collagen were constructed both in the absence and presence of 2 microM SIN-1, an NO donor, to quantify the antiaggregation effects of NO. After exercise, platelet aggregation by collagen was modestly enhanced and inhibition of platelet aggregation by SIN-1 was significantly attenuated by exercise. This attention was accompanied by a blunted cyclic GMP response of the platelets to the NO donor. We conclude that impaired sensitivity of the platelets to NO, in addition to the enhancement of platelet aggregation, may partially explain the epidemiological evidence that acute strenuous exercise precipitates coronary events.

The induction by topical minoxidil of increased fenestration in the perifollicular capillary wall.

We report the induction by topical minoxidil of increased fenestration in the perifollicular capillary wall. Male 30-day-old Wistar rats were divided into two groups, i.e. an experimental group which received 4% minoxidil solution topically on the dorsal skin, and a control group which received only vehicle solution topically. Using transmission electron microscopy, follicular and subepidermal capillaries and dermal fibres were compared between both groups. There were no obvious differences in subepidermal capillaries or dermal fibres between the two groups. However, topically applied minoxidil increased fenestration in follicular capillary walls around anagen hair bulbs.

Electron microscopic characterization of filiform papillae in the normal human tongue.

The fine structure of filiform papillae on the normal human tongue was examined level by level, from the basal layer to the surface, in specimens taken from the dorsal side of the lingual body. Human lingual epithelia showed three distinct regions: epithelia on the anterior and on the posterior sides of filiform papillae and an interpapillary epithelium. While the basal and the squamous cell layers were similar throughout these three regions, differences were noted in the granular and the horny layers. The interpapillary epithelium actually lacked both the granular and the horny layers. The epithelium on the anterior side of filiform papillae was characterized by alternating layers of granular cells and of cornified cells. Granular cells possessed three types of keratohyaline-like granules within their cytoplasm: uniformly electron dense, relatively less electron dense, and a heterogeneous type. While the number of the keratohyaline-like granules was remarkably diminished in the epithelium on the posterior side of filiform papillae, a considerable amount of tonofibrils was present in the cytoplasm. In the uppermost portion of the anterior side of filiform papillae, coherence between adjacent epithelial cells depended mainly on the interlocking of cytoplasmic villi and poorly developed desmosomes on villi. On the other hand, epithelial cells on the posterior side of filiform papillae appeared to be more tightly adhesive compared with those on the anterior side. This was due to focal thickening of the plasma membrane and to desmosomes at the interface between the granular and cornified cells, and to the formation of a marginal band and increased intercellular cement presumably derived from lamellar bodies in the horny layer. These findings demonstrate distinct differences between the anterior and the posterior sides of filiform papillae in the human tongue with respect to keratinization patterns, structures associated with cell-to-cell adhesion and the strength of cellular cohesion in the uppermost portion, and the turnover of cornified cells. These differences may contribute to the formation of the unique external configurations of filiform papillae.

Effect of nifedipine on cyclic GMP turnover in cultured coronary smooth muscle cells.

We investigated the effects of nifedipine on cyclic GMP turnover and the pertinent enzyme activities in cultured coronary smooth muscle cells (SMC). Nifedipine at high concentrations slightly decreased basal soluble guanylate cyclase activity and inhibited the action of sodium nitroprusside (SNP) but had no effect on the particulate form of the enzyme. In contrast, nifedipine inhibited cyclic GMP hydrolysis by directly inhibiting the partially purified calmodulin-stimulated isoform of phosphodiesterase (type I PDE) with IC50 of 4.2 microM. Nifedipine > or = 1.0 microM enhanced cyclic GMP accumulation in response to 1.0 microM SNP, although nifedipine alone exerted no influence on cyclic GMP levels. Enhancement of cyclic GMP accumulation by nifedipine in response to SNP was not affected by BAY K 8644, a calcium channel agonist. These properties may be shared by other dihydropyridines since nicardipine and nisoldipine also inhibited type I PDE with similar IC50. However, some other structurally unrelated calcium channel blockers, diltiazem and verapamil, had little effect on cyclic nucleotide hydrolysis or on cyclic GMP accumulation in response to SNP. Nifedipine may synergistically enhance cyclic GMP accumulation in response to nitric oxide (NO)-releasing agents by directly inhibiting type I PDE in coronary SMC. Such effects of nifedipine may partly contribute to coronary vasodilation and prevention of coronary spasm in patients with ischemic heart disease.

Ibudilast: a non-selective PDE inhibitor with multiple actions on blood cells and the vascular wall.

Ibudilast (3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine) is a nonselective inhibitor of cyclic nucleotide phosphodiesterase (PDE). It is widely used in Japan for improving prognosis and relieving symptoms in patients suffering from ischemic stroke or bronchial asthma. These clinical applications are based on the properties of ibudilast that inhibit platelet aggregation, improve cerebral blood flow and attenuate allergic reactions. The inhibition of platelet aggregation and vasodilatation by ibudilast may be due to synergistic elevation of intracellular cyclic nucleotides and release of nitric oxide (NO) or prostacyclin from endothelium, rather than direct inhibition of PDE5 or PDE3. Another important property of ibudilast is its antiinflammatory activity possibly associated with potent inhibition of PDE4. Combined with its relaxing effects on bronchial smooth muscle, antiinflammatory activity of ibudilast could favorably influence pathophysiology of asthma by antagonizing chemical mediators triggering asthmatic attacks. Ibudilast was also reported to significantly attenuate inflammatory cell infiltration in the lumbar spinal cord in an animal model of encephalomyelitis. Future investigations should include effects of ibudilast on inflammatory reactions between endothelium and blood cells, which may initiate the development of atherosclerosis.

Ibudilast modulates platelet-endothelium interaction mainly through cyclic GMP-dependent mechanism.

3-Isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast) has been widely used in Japanese clinics for its antiasthmatic and antithrombotic effects. We investigated the mechanisms involved in the antiplatelet effects of the agent, specifically focusing on platelet-endothelium interaction. Ibudilast inhibits both phosphodiesterase (PDE) 3 and 5, the two major PDE isoforms of human platelets, with an IC50 of 31 and 2.2 microM, respectively. Cyclic guanosine monophosphate (GMP) accumulation in washed human platelets exposed to ibudilast alone increased significantly only at high concentrations of the agent (100 microM), whereas > or = 1 microM ibudilast enhanced cyclic GMP levels in the platelets cocultured with bovine aorta endothelial cells (ECs). In contrast, ibudilast enhanced cyclic AMP accumulation only at 100 microM, either with or without ECs. The synergistic effect of ibudilast and EC on cyclic nucleotide accumulation also was demonstrated by the inhibitory capability of the drug and the cells on platelet aggregation. The synergism between ibudilast and aspirin-pretreated ECs was more pronounced than that between ibudilast and N(omega)-nitro-L-arginine (L-NNA)-pretreated ECs. Ibudilast affected neither ATP diphosphohydrolase activity nor NO release from EC up to a concentration of 10 microM. We conclude that ibudilast exhibits antiplatelet properties mainly by inhibiting PDE5 to potentiate antiplatelet function of endothelium-derived NO.