Increase of metabolic activity and disruption of normal contractile protein distribution by bilirubin oxidation products in vascular smooth-muscle cells.

OBJECT: Cerebral vasospasm is a common cause of morbidity and death following aneurysmal subarachnoid hemorrhage (SAH). Previous research has shown that bilirubin oxidation products (BOXes) are present in the cerebral spinal fluid in patients with SAH-induced cerebral vasospasm and can contribute to vasoconstriction and vasospasm in vitro and in vivo. The events leading to cerebral vasospasm are not understood; however, one component of the occlusion may be due to vascular remodeling. In this study the authors have investigated the actions of BOXes, okadaic acid ([OA], a phosphatase inhibitor), and phorbol-12 myristate-13 acetate ([PMA], a protein kinase activator) on vascular smooth-muscle cell (VSMC) morphology and metabolism. METHODS: Immunohistochemical analysis was performed to assess VSMC morphology and alpha-smooth-muscle actin (alphaSMA) distribution following the application of BOXes, OA, or PMA. Changes in the level of lactate dehydrogenase (LDH) release and oxidative metabolism were also measured. The BOXes, OA, or PMA caused VSMCs to change their shape and exhibit altered alphaSMA distribution. These treatments increased LDH release (p < 0.05), which is an index of increased cell stress. Oxidative metabolism significantly increased at low and high doses of BOXes, that is, 143 +/- 8.5% and 180 +/- 11.8%, respectively (p < 0.0001). Both PMA and OA also caused a significant increase in metabolism. CONCLUSIONS: The authors concluded that BOXes, OA, and PMA alter VSMC morphology and metabolic activity, events that have been observed during vascular remodeling. Although the mechanism remains unclear, the results indicate that BOXes may play a role in the vascular remodeling that occurs following aneurysmal SAH.

Platelets play an essential role in the aetiology of cerebral vasospasm after subarachnoid haemorrhage.

Platelets have long been implicated in the aetiology of cerebral vasospasm (CV) after subarachnoid haemorrhage (SAH). It was noticed that vasospastic CSF (CSF(V)) could be formed in vitro by the mixing of control blood (with platelets) and non-SAH CSF. We also propose a hypothesis for the aetiology of CV after SAH based on this and previous research. This study also aims to determine which blood fraction is responsible for the stimulation of O(2) consumption and vasospasm of blood vessels. Control blood was separated into various fractions and mixed with non-SAH CSF. The activity of the resulting mixture and the blood fraction alone were assessed. Only the fractions containing platelets mixed with CSF showed vasoactivity. These data suggest that platelets plus some component in the CSF produce vasoactive factors with actions similar to CSF(V). This study may help to elucidate the aetiology of CV after SAH.

Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins.

We previously generated an isoform-specific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogen-activated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC(17b)) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins.

Targeted ablation of plasma membrane Ca2+-ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4.

The relative importance of plasma membrane Ca2+-ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4-/-) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4-/- mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4-/- male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4-/- sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4-/- sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca2+ overload. Immunoblotting and immunohistochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca2+ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.