Platelet lipoxygenase inhibitors attenuate thrombin- and thromboxane mimetic-induced intracellular calcium mobilization and platelet aggregation.

Platelets metabolize arachidonic acid via cyclooxygenase and lipoxygenase (LO) enzymatic pathways. Although platelets produce large amounts of arachidonic acid metabolites via the LO pathway, little is known regarding the physiological significance of these products. We used three structurally dissimilar LO inhibitors, 5,8,11-eicosatriynoic acid (ETI), baicalein and phenidone, and found that LO inhibition attenuated thrombin- and U46619 (a thromboxane mimetic)-induced increases of platelet intracellular calcium ([Ca++]i) in washed human platelets. LO inhibitors also reduced platelet aggregation induced by thrombin and U46619. The effect of ETI on reducing the thrombin-induced [Ca++]i elevation persisted even when cation channels were blocked, suggesting that LO inhibitors modify release of Ca from intracellular stores. Stimulating endogenous LO product formation potentiated thrombin-induced [Ca++]i responses and aggregation, and these effects were eliminated by ETI. ETI did not alter inositol 1,4,5-trisphosphate production in stimulated platelets, but increased platelet cyclic AMP production in thrombin- or forskolin-stimulated platelets. These results suggest that LO products are regulators of platelet [Ca++]i mobilization and aggregation in response to some agonists, and that LO inhibitors may work in part by modifying platelet cyclic AMP metabolism.

Desensitization of vascular tissue to parathyroid hormone and parathyroid hormone-related protein.

Although PTH and PTH-related protein (PTHrP) are vasodilators, prolonged exposure to elevated levels of PTH is often associated with hypertension. We investigated the effects of prolonged incubation with PTH or PTHrP on arterial segments and cultured vascular smooth muscle cells (VSMC). PTH or PTHrP transiently relaxed precontracted arterial segments within 10 min. Additional PTH or PTHrP added after 40-min exposure to these peptides had little effect on vascular tone, whereas forskolin, isoproterenol, isobutylmethyl-xanthine, or acetylcholine were still potent. In fura 2-loaded VSMC, 5-min incubation with PTH or PTHrP attenuated angiotensin II (Ang II)-induced calcium mobilization, an effect that was reduced by preincubation of VSMC with PTH for 1.5 h. Similarly, 1.5-h preincubation with PTH or PTHrP decreased the cAMP response to these peptides but not to forskolin or NaF. Ang II potentiated the cAMP response to PTH and PTHrP but was also subject to desensitization. Nle8, 18Tyr34 bovine PTH(3-34) amide did not desensitize vascular tissue to PTH or PTHrP. Our results suggest that homologous desensitization to PTH or PTHrP in vascular tissue requires receptor stimulation, occurs proximal to G stimulatory protein, and impairs attenuation of calcium mobilization by PTH or PTHrP. This may be a mechanism by which vasodilator effects of these peptides are decreased with prolonged elevation of PTH levels.

Parathyroid hormone analogues inhibit calcium mobilization in cultured vascular cells.

Parathyroid hormone and parathyroid hormone-related protein lower blood pressure and relax contracted arteries. Parathyroid hormone also attenuates angiotensin II-induced vasoconstriction. To determine the cellular mechanism or mechanisms by which parathyroid hormone analogues antagonize pressor effects, we examined the effect of these peptides on angiotensin II-induced calcium mobilization in fura 2-AM-loaded cultured rat vascular smooth muscle cells. Either 100 nmol/L parathyroid hormone or parathyroid hormone-related protein significantly reduced the amount of calcium mobilized by 100 nmol/L angiotensin II. The attenuating effect of these peptides was mimicked by 10 mmol/L forskolin and 10 mmol/L isobutylmethylxanthine and was not dependent on the presence of extracellular calcium. This effect of the parathyroid hormone analogues was reduced when cells were pretreated with 100 mmol/L 2',5'-dideoxyadenosine, an adenylate cyclase inhibitor. Combined inhibition of cyclic nucleotide-dependent protein kinases eliminated the inhibitory effect of parathyroid hormone, whereas protein kinase C inhibition had no effect. Parathyroid hormone analogues decreased the amount of calcium released by inositol 1,4,5-trisphosphate in digitonin-permeabilized vascular smooth muscle cells. This effect was inhibited by treatment with 2',5'-dideoxyadenosine. These results suggest that these peptides attenuate inositol 1,4,5-trisphosphate-sensitive calcium mobilized by angiotensin II via an adenylate cyclase-dependent mechanism. This may be a mechanism by which acute administration of parathyroid hormone or parathyroid hormone-related peptide antagonizes vasoconstriction.

Racial differences in platelet cytosolic calcium and calcitropic hormones in normotensive subjects.

Elevated levels of serum parathyroid hormone (PTH) and platelet cytosolic free calcium ([Ca2+]i) have been reported in subjects with essential hypertension. In addition, there is a positive correlation between serum PTH and platelet [Ca2+]i in white subjects with essential hypertension. Black normotensive subjects have relatively higher levels of serum PTH when compared to white normotensive subjects. To investigate the possibility that elevated serum PTH levels in black normotensives may contribute to elevated platelet [Ca2+]i, calcitropic hormone profiles and platelet [Ca2+]i were determined in 31 black normotensive subjects and 34 age-matched white normotensive subjects. There was no difference between the two groups in total serum calcium, plasma ionized calcium, or creatinine clearance. However, serum PTH was significantly elevated (P < .02) in the black normotensive group. Serum 1,25(OH)2 vitamin D levels were similar between the two groups whereas serum 25(OH) vitamin D levels were significantly lower (P < .001) in the blacks. The 24 h urinary excretion of Ca was also lower (P < .05) in the black normotensive group. Basal platelet [Ca2+]i was significantly lower (P < .05) in black normotensive than in white normotensive subjects. Serum PTH levels did not correlate with platelet [Ca2+]i in either group, or in the groups combined. These results demonstrate that the higher serum PTH concentrations in black normotensives is not associated with higher platelet [Ca2+]i, as is the case in white hypertensive patients.

Flavonoid potentiation of contractile responses in rat blood vessels.

Certain bioflavonoids and phenolic compounds have long been known to enhance catecholamine responses, in vivo and in vitro. In the present studies the flavone, baicalein, potentiated nerve-stimulated contractions in vitro in rat tail and femoral artery isometric ring preparations. Inhibition of catecholamine reuptake with cocaine or catecholamine metabolism with tropolone and parglyine (monoamine oxidase and catecholamine-O-methyl transferase inhibitors, respectively) did not alter baicalein's ability to potentiate contractile responses to nerve stimulation. Baicalein (10(-5) M), the prototype flavone, also increased sensitivity to exogenous norepinephrine, serotonin, arginine vasopressin and to the noncatecholamine alpha-1 and alpha-2 adrenergic agonists, cirazoline and tramazoline. Structure-function studies indicated that flavone potentiation required three contiguous A or B ring hydroxylations. Several nonflavone phenol derivatives with three contiguous hydroxyls also potentiated nerve stimulation responses. As baicalein is a potent lipoxygenase inhibitor, comparisons were made between potentiating ability and lipoxygenase inhibitory activity in a series of flavonoids. There was no direct correlation between inhibition of 12-hydroxy-5,8,10,14-eicosatetraenoic acid levels in thrombin stimulated human platelets and potentiation of contractile responses in the femoral artery. Additionally, the specific substrate analog lipoxygenase inhibitor, 5,8,11-eicosatriynoic acid, and the cyclooxygenase inhibitor, ibuprofen, were nonpotentiating. Ibuprofen pretreatment did not alter the potentiating action of baicalein. It is concluded that flavonoids with three contiguous hydroxyls on either the A or B ring increase in vitro vascular responsiveness via a post-synaptic process, independent of cyclooxygenase, lipoxygenase, monoamine oxidase or catecholamine-O-methyl transferase activity.

Calcitropic hormones, platelet calcium, and blood pressure in essential hypertension.

Plasma ionized calcium, platelet cytosolic calcium (using the fura-2 method in gel-filtered platelets), parathyroid hormone (both the intact hormone and a midmolecule portion), calcitriol, and calcidiol were measured in 19 untreated male patients with essential hypertension and 19 age-matched normotensive male research subjects. Mean levels of platelet cytosolic calcium, parathyroid hormone, calcitriol, and calcidiol were all significantly higher, whereas plasma ionized calcium was significantly lower, in the hypertensive group compared with the normotensive group. Both platelet cytosolic calcium and intact parathyroid hormone were positively correlated with mean arterial pressure (r = 0.58, p less than 0.001; r = 0.54, p less than 0.001, respectively), whereas plasma ionized calcium was inversely correlated with mean arterial pressure (r = -0.60, p less than 0.001) in the combined group of all study subjects. All three of these correlations were significant in the hypertensive group alone but not in the normotensive group alone. When analyzed with plasma ionized calcium, body mass index, serum calcitriol, and calcidiol in a multivariable regression model, the significance of the partial regressions of platelet cytosolic calcium and parathyroid hormone with mean arterial pressure persisted. Intact parathyroid hormone was positively correlated to platelet cytosolic calcium (r = 0.43, p less than 0.01) and plasma ionized calcium was inversely correlated to platelet cytosolic calcium (r = -0.44, p less than 0.01). These results confirm previous reports of disturbances of calcium metabolism in essential hypertension and suggest that the elevated platelet cytosolic calcium observed in essential hypertension may be linked to one or more of these alterations of calcium metabolism.

Decreased vascular reactivity to norepinephrine in Fischer rats with neoplasia-induced hypercalcemia.

The effect of a hypercalcemia-producing Leydig cell tumor on vascular reactivity in Fischer rats was studied. Seven to eight days after tumor implantation, there was no difference between tumor (T) and control (C) animals in serum calcium, serum phosphate, plasma catecholamine levels, mean arterial pressure (MAP), or blood pressure responses to norepinephrine (NE) infusion. At day 12-13 of tumor growth, the serum calcium in the tumor-bearing rats was significantly higher (12.2 +/- 0.8 vs. 9.7 +/- 0.3 mg%, P less than .01) and their serum phosphate significantly lower (4.5 +/- 0.3 vs. 5.7 +/- 0.4 mg%, P less than .01) than controls. Plasma epinephrine (E) (497 +/- 154 vs. 62 +/- 13 pg/ml, P less than .05), and norepinephrine (NE) (686 +/- 85 vs. 329 +/- 75 pg/ml, P less than .01) were markedly elevated in the tumor rats. MAP and the blood pressure responses to graded NE infusions were significantly lower in tumor animals at Day 12-13, whereas there was no change in sensitivity to angiotensin II (AII) infusions. In vitro contractile responses of tail artery segments to transmural nerve stimulation (TNS) in animals with tumors were lower than in controls but there were no differences in sensitivity to exogenous NE in vitro. These results suggest that the tumor stimulates production of a circulating factor which desensitizes NE receptors and that this tumor also decreases neurovascular function by an undefined mechanism.

Isolation, characterization, and numerical taxonomy of Simonsiella strains from the oral cavities of cats, dogs, sheep, and humans.

Forty-nine strains of the gliding prokaryote Simonsiella were isolated from the oral cavities of cats (8), dogs (19), sheep (4), and humans (18) in Southern California by a direct isolation procedure using a complex serum-enriched medium. The numerical taxonomic analysis (unweighted pair-group method using arithmetric averages) of 57 differential traits for each strain was based on standard bacteriological diagnostic tests and included the molar guanine-plus-cytosine contents of the DNA and the relative percentages of fatty acid contents reported earlier. The resulting phenogram clustered the strains of Simonsiella into groups that correlated with sources of origin. The study included the neotype strain of Simonsiella crassa (ATCC 27504, ICPB 3651, NCTC 10283) of Australian sheep origin. The strains isolated from dogs, sheep, and humans form clusters of organisms that appear to have become adapted to live in and possibly to have evolved with their respective "hosts". In our judgment, these source-of-origin clusters represent different "ecospecies".

Incidence of Simonsiella in the oral cavity of dogs.

The gliding bacterium Simonsiella (Cytophagales, Simonsiellaceae) was found in palate samples from 66 out of 67 dogs. It is considered a common resident in the oral cavities of dogs.

Prostaglandin synthesis inhibition restores hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction in blood-perfused isolated dog lungs progressively diminishes with repeated hypoxic challenges. We investigated the role of prostaglandins in effecting the decay of the hypoxic response by using a double perfusion preparation that could separately perfuse the right and left lungs of a single dog. Degeneration of this response was reversed by the addition of prostaglandin (PG) synthesis inhibitors, aspirin, or indomethacin. Various PG's known to be produced by the lung (PGE1, PGE2, and PGF2alpha), were infused, and only PGE1 abolished hypoxic pulmonary vasoconstriction. Since other workers have shown that lungs can synthesize and release PG's in response to various stimuli, we postulate that PGE1 synthesis in isolated lungs may increase and thereby cause the degeneration of the hypoxic response. The addition of aspirin or indomethacin could inhibit the synthesis of PGE1 and thereby restore hypoxic pulmonary vasoconstriction.

Deoxyribonucleic acid base composition of Simonsiellaceae.

The molar percentages of guanine plus cytosine in the DNA of 51 strains of Simonsiellaceae were determined by buoyant density ultracentrifugation of cell lysates in CsCl. The DNA base ratios ranged from 41-55 mole-% guanine plus cytosine. These values fall within the range known for the Order Cytophagales, the non-fruiting gliding bacteria, and are outside the range of the Order Myxobacterales, the fruiting myxobacteria. Among the strains of the genus Simonsiella, four distinct group can be delineated on the basis of source of origin (sheep, dog, cat, human) and GC content. The neotype of Alysiella filiformis has a GC content of 45.4 mole-%.

Effect of angiotensin on hypoxic pulmonary vasoconstriction in isolated dog lung.

The effects of infused angiotensin on hypoxic pulmonary vasoconstriction in blood-perfused isolated dog lungs were studied. By using a double-perfusion system we were able to perfuse the right and left lungs separately in the same animal; one lung was used as control and the other lung was experimentally modified. The vasoconstrictive response to hypoxia decreased with time in the isolated lung preparations. The infusion of either angiotensin I or angiotensin II (1.2-5.8 mug/min) caused a threefold increase in the vasoconstrictive response to hypoxia over control levels. A second hypoxic period during the infusion usually yielded a diminished response, suggesting further degeneration of the response irreversible with angiotensin. It was concluded that angiotensin I or angiotensin II temporarily enhances hypoxic pulmonary vasoconstriction in isolated dog lungs.