Bozena Vollrath

Functional Roles of the Rho/Rho Kinase Pathway and Protein Kinase C in the Regulation of Cerebrovascular Constriction Mediated by Hemoglobin. Relevance to Subarachnoid Hemorrhage and Vasospasm

Although there is evidence that the Rho/Rho kinase pathway and protein kinase C (PKC) are involved in the development of cerebral vasospasm, the mechanism by which subarachnoid hemorrhage (SAH) activates these pathways is unclear. A large body of evidence points to oxyhemoglobin (OxyHb) as a major causative component of blood clot responsible for vasospasm. Therefore, the present studies were conducted to explore whether the Rho/Rho kinase and PKC may be involved in a sustained vasoconstriction induced by OxyHb in cerebral arteries. OxyHb evoked sustained vasoconstriction in the endothelium-denuded rabbit basilar arteries, which was reversed by the selective inhibitors of Rho kinase, Y-27632, and HA-1077, with the IC50 values of 0.26+/-0.02 and 0.74+/-0.1 micromol/L, respectively. In quiescent cerebrovascular smooth muscle (CVSM) cells, OxyHb induced Rho translocation, as assessed by immunoblotting, with a time course, which paralleled the contractile action of OxyHb. Rho translocation was also observed in intact arteries stimulated with OxyHb for 24 hours (219%) and 48 hours (160%). The increase in Rho translocation was fully inhibited by GGTI-297, an inhibitor of Rho prenylation. OxyHb also caused significant translocation of both PKCalpha and PKCepsilon (P<0.01), which was maximal at the time corresponding to maximal tension developed in response to OxyHb. Ro-32-0432, an inhibitor of PKC, attenuated vasoconstriction mediated by OxyHb in basilar artery. These results show, for the first time, that OxyHb-mediated signaling in CVSM utilizes the Rho/Rho kinase and PKC-based mechanisms.

Effects of statins on vascular function of endothelin-1.

1 Although statins have been reported to inhibit the prepro‐endothelin‐1 (ET‐1) gene transcription in endothelial cells, their effects on the vascular function of ET‐1 have not been explored. We, therefore, examined the effects of statins on contraction and DNA synthesis mediated by ET‐1 in vascular smooth muscle. The effects of statins on contraction induced by ET‐1 were compared to those mediated by noradrenaline (NA) and KCl. 2 Simvastatin (SV) induced a concentration‐dependent relaxation of tonic contraction mediated by ET‐1 (10 nM) (IC50 value of 1.3 μM). The relaxation was also observed in rings precontracted with NA (0.1 μM) and KCl (60 mM). In contrast, pravastatin did not have any effect on the contractions. 3 Endothelial denudation or pretreatment with L‐NAME did not prevent the relaxation, but did reduce the relaxant activity of SV. 4 SV prevented Rho activation caused by ET‐1 and KCl in aortic homogenates, as assessed by a Rho pulldown assay. 5 The Rho kinase inhibitor HA‐1077 mimicked the effects of SV on tonic contractions induced by ET‐1, NA and KCl. 6 Pretreatment with the Kv channels inhibitor, 4‐aminopyridine, attenuated the ability of SV to relax contractions mediated by ET‐1 and NA. 7 In quiescent VSM cells, SV significantly inhibited DNA synthesis and Rho translocation stimulated by ET‐1, as assessed by [3H]thymidine incorporation and Western blot, respectively. 8 Inhibition of Rho geranylgeranylation by GGTI‐297, or treatment with HA‐1077, mimicked the effects of SV on DNA synthesis stimulated by ET‐1. 9 The results show that the statin potently inhibits both ET‐1‐mediated contraction and DNA synthesis via multiple mechanisms. Clinical benefits of statins may result, in part, from their effects on vascular function of ET‐1.

Time course of changes in concentration of intracellular free calcium in cultured cerebrovascular smooth muscle cells exposed to oxyhemoglobin.

A culture of smooth muscle cells obtained from monkey middle cerebral arteries was developed to allow quantitative assessment of intracellular calcium and immunofluorescence analysis after various periods of exposure to oxyhemoglobin. Intracellular calcium concentration was examined for up to 7 days after a single exposure to oxyhemoglobin. Intracellular calcium concentrations were measured with the fluorescent dye fura-2 and were significantly elevated for 7 days after exposure to oxyhemoglobin (P less than 0.01). Less than 2 minutes after application of oxyhemoglobin, there was marked elevation of intracellular calcium from the control value of 75 +/- 2 nmol/L to 240 +/- 28 nmol/L (P less than 0.01 by analysis of variance). Intracellular calcium concentration of cells exposed for 24 hours to oxyhemoglobin and then grown in normal oxyhemoglobin-free medium fell close to normal levels on Days 3 and 7. On Day 3, the increase in intracellular calcium that followed repeated daily exposure to oxyhemoglobin was greater than that resulting from a single application of oxyhemoglobin (P less than 0.01 by Students t test), but by Day 7 the elevation produced by these different approaches was similar. Smooth muscle cells exposed to oxyhemoglobin showed a reduction in immunoreactivity to alpha-actin. These data support the hypothesis that disruption of intracellular calcium regulation and calcium overloading may be important in the process of cell injury, which results in vasoconstriction and sometimes cell death, after exposure to oxyhemoglobin.

Novel mechanism by which hemoglobin induces constriction of cerebral arteries

Since oxyhemoglobin (OxyHb) is implicated in the pathogenesis of cerebral vasospasm, we have investigated the role of protein tyrosine phosphorylation in OxyHb-mediated signalling in canine cerebral arteries and cultured canine cerebrovascular smooth muscle cells. OxyHb produced a contraction of basilar artery preparations, which was reversed by genistein, an inhibitor of tyrosine kinases, and PD098059, an inhibitor of mitogen-activated protein kinase. In cerebrovascular smooth muscle cells, OxyHb induced tyrosine phosphorylation of 42, 46, 54-60 and 80-100 kDa proteins with a time-course which paralleled the contractile action of OxyHb, suggesting that these events might be functionally linked. The 42 and 60 kDa proteins were immunologically related to the mitogen-activated protein kinase, extracellular signal regulated protein kinase (ERK2), and to p60c-Src (c-Src), respectively. The increase in protein tyrosine phosphorylation was attenuated by genistein, and the phosphorylation of the 42 kDa protein (ERK2) was inhibited by PD098059. These results suggest that OxyHb-mediated signalling utilizes a protein tyrosine kinase-based mechanism.

Procollagen types I and III and transforming growth factor-beta gene expression in the arterial wall after exposure to periarterial blood.

The stiffening and thickening of the arterial wall after subarachnoid hemorrhage may reflect increased connective tissue. The purpose of this study was to examine the nature of collagen synthesis in response to periarterial blood. Rat femoral arteries were exposed to periarterial blood for varying lengths of time (control, 1, 3, 7, and 14 d). Dot-blot analysis of total ribonucleic acid extracted from the arteries (n = 10 to 15 animals each) demonstrated that the expression of procollagen Types I and III messenger ribonucleic acid increased at 7 (threefold) and 14 days. The expression of transforming growth factor-beta (TGF-beta), an important regulator of collagen synthesis, was markedly increased by 3 days (threefold), followed by a gradual decline. There were marked differences in procollagen Types I and III and TGF-beta gene expression between arteries exposed to blood and sham-operated arteries for a period of 7 days (n = 25 animals). Northern blot analysis of total ribonucleic acid extracted from cultured vascular smooth muscle cells showed that the treatment with a higher concentration of serum for 48 hours increased the expression of procollagen Types I and III and TGF-beta, whereas exposure to oxyhemoglobin did not. After exposure to periarterial blood, arterial walls show increased synthesis of procollagen Types I and III, perhaps a response to the increased secretion of TGF-beta, which in turn could be the result of exposure to serum factors.

Vasodilatation of canine cerebral arteries by nicorandil, pinacidil and lemakalim

1. Nicorandil, pinacidil and lemakalim relaxed precontracted rings of canine cerebral artery. 2. The order of potency was lemakalim greater than nicorandil approximately equal to pinacidil, but all these agents were less effective than nimodipine. 3. The effects of nicorandil were inhibited by methylene blue but not by glibenclamide, while the effects of pinacidil and lemakalim were inhibited by glibenclamide but not by methylene blue. 4. Thus nicorandil probably causes relaxation mostly by effects on guanylate cyclase while lemakalim and pinacidil produce the same effect by action at ATP-dependent potassium channels.

The mechanism by which aminoglycoside antibiotics cause vasodilation of canine cerebral arteries

1 The effects of aminoglycoside antibiotics were examined in canine cerebral arteries and in cultured cerebrovascular smooth muscle cells stimulated with oxyhemoglobin (OxyHb), a blood constituent which has been implicated in the pathogenesis of cerebrovascular spasm. 2 In cerebral arterial rings precontracted with OxyHb (10 μm), the aminoglycosides caused a concentration‐dependent decrease in isometric tension. The EC50s for the relaxation were 0.46±0.1 mm (n = 6), 0.53±0.08 mm (n = 12), 1.6±0.3 mm (n = 7) and 3.9±0.5 mm (n = 5) for neomycin, gentamicin, streptomycin and kanamycin, respectively. This order of potency corresponds approximately to the number of positive charges in the molecules. 3 The aminoglycosides also inhibited the contractions to prostaglandin F2α (1 μm) and depolarizing concentrations of potassium chloride (60 mm). The order of potency was neomycin>gentamicin>streptomycin>kanamycin. 4 The relaxation was maintained in vascular preparations denuded of endothelium. 5 Neomycin (5 mm) abolished the Ca2+‐independent contraction to PGF2α. 6 In Fura‐2‐loaded cerebrovascular smooth muscle cells, OxyHb (1 μm) significantly enhanced the concentration of intracellular calcium ([Ca2+]i) by 330%. The administration of neomycin, gentamicin, kanamycin and streptomycin in concentrations corresponding to the EC50 from contractility studies, reduced the effects of OxyHb on [Ca2+]i by about 50% to 221±35 nm (n = 7), 270±31 nm (n = 7), 229±33 nm (n = 6) and 240±6 nm (n = 5), respectively. 7 These results suggests that the effects of the aminoglycosides on the OxyHb‐induced contraction and the long‐term increase in [Ca2+]i, may arise from several effects, including inhibition of PLC, protection of calcium extrusion mechanisms, and interference with the process of [Ca2+]i accumulation.

Hemoglobin causes release of inositol trisphosphate from vascular smooth muscle

To test the hypothesis that oxyhemoglobin causes contraction of vascular smooth muscle by production of inositol 1,4,5-trisphosphate which results in a release of intracellular calcium, smooth muscle cells were exposed to oxyhemoglobin and inositol trisphosphate was measured. Oxyhemoglobin, but not methemoglobin which has much less contractile action, stimulated inositol trisphosphate production. The time course was consistent with an early role for this compound in the contraction produced by hemoglobin. The increase in production of inositol trisphosphate was inhibited by pertussis toxin and also by neomycin, an inhibitor of phospholipase C, although the actions of the latter compound cannot be attributed only to an inhibition of the enzyme responsible for the production of inositol trisphosphate.

The polycationic aminoglycosides modulate the vasoconstrictive effects of endothelin: relevance to cerebral vasospasm

The vasoactive peptide endothelin (ET) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid haemorrhage. In these studies we investigated the involvement of protein kinase C (PKC) in sustained vasoconstriction induced by ET‐1 in canine cerebral arteries. We also examined the ability of the aminoglycoside antibiotics to reverse the effects mediated by ET‐1 in canine cerebrovascular smooth muscle cells (CVSMC). The ETA receptor antagonist, BQ‐123, showed a competitive inhibition of the ET‐1 responses. The vasoconstrictor action of both ET‐1 (0.5 nM) and phorbol myristate acetate (PMA) (160 nM) was reversed by a selective PKC inhibitor, Ro‐32‐0432. In cerebral arteries precontracted with ET‐1 the aminoglycosides caused a concentration‐dependent relaxation. The EC50s for the relaxation were as follows: 0.54±0.05, 0.63±0.01, 1.88±0.46 and 2.3±0.92 mM for gentamicin, neomycin, streptomycin and kanamycin, respectively. Gentamicin caused a concentration‐dependent decrease of the PMA‐induced responses in calcium free medium. PKC activity was elevated in CVSMC exposed to ET‐1 (170%) and PMA (167%) for a period of time (60 min) corresponding to maximum tonic contraction induced by these agents in arterial rings. The administration of the aminoglycosides to CVSMC, in concentrations corresponding to the EC50s from contractility studies, reduced the effects of both ET‐1 and PMA on PKC activity to the levels not different from controls. These results show that the aminoglycosides are able to inhibit sustained vasoconstriction induced by ET‐1, an effect which is due, at least in part, to the inhibition of PKC.

Effects of tamoxifen on oxyhemoglobin-induced cerebral vasoconstriction

The effect of tamoxifen on oxyhemoglobin-mediated cerebral vasoconstriction was examined. Tamoxifen caused a concentration-dependent relaxation of cerebral artery preparations contracted with oxyhemoglobin and phorbol myristate acetate with the IC(50) values 0.66+/-0.1 and 1.1+/-0.1 microM, respectively. In cerebrovascular smooth muscle cells, oxyhemoglobin and phorbol myristate acetate induced protein kinase C activation, which was 220+/-7% and 203+/-8% of control, respectively. The increase in protein kinase C activity was prevented by tamoxifen. The results suggest that the ability of tamoxifen to reverse vasoconstriction is mediated, at least in part, via inhibition of protein kinase C.