Isolation of anaerobic, extremely thermophilic, sulphur metabolising archaebacteria from New Zealand hot springs

Enrichments of New Zealand geo-thermal samples, initiated in anaerobic sulphur-containing media and incubated at temperatures above 85°C, yielded rod and coccal shaped organisms which possessed archaebacterial characteristics. Pure cultures were isolated and characterised. Five of the seven isolates, which were rod-shaped organisms and did not have an obligate requirement for sulphur respiration, were similar to Ther-moproteus sp. but had more neutral pH optima for growth. Three of these five Thermoproteus sp. were obligate heterotrophs, which has not previously been reported. The two coccal isolates had an obligate requirement for sulphur as an electron acceptor and were similar to Desulfurococcus sp. but again with more neutral pH optima for growth

Mechanism of nitric oxide induced sympatholysis in rat soleus feed arteries

During exercise, the neurotransmitter norepinephrine (NE) binds to arterial adrenergic receptors to cause vasoconstriction, yet arteries and arterioles constrict less to sympathetic stimulation in contracting compared to resting skeletal muscle (sympatholysis). Previous evidence indicates that nitric oxide (NO) can be sympatholytic, but the mechanism is unknown. We hypothesized that NO causes sympatholysis in rat soleus muscle feed arteries, that NO is released from vascular endothelial cells by increased shear stress, and that NO acts through a guanylyl cyclase intracellular signaling pathway. Soleus feed arteries (n = 12 per group) were isolated from male Sprague-Dawley rats and cannulated on two glass micropipettes for in vitro videomicroscopy. We measured the constriction response to the adrenergic agonist phenylephrine (PE; 10-9 M to 10-4 M, 0.5 log increments) in the presence of varying levels of the nitric oxide donor sodium nitroprusside (SNP; 0 nM, 0.1 nM and 100 nM), shear stress (0 dy/cm2, 25 dy/cm2, and 135 dy/cm2), and SNP + ODQ (0.1 nM), an inhibitor of guanylyl cyclase. SNP reduced constriction to PE in a dose-dependent manner (maximum constriction 77.3 % vs. 70.7 % and 56.7 %), indicating that NO interferes with sympathetic constriction. ODQ restored PE-induced constriction (PE alone 77.5%; with SNP 67.6%; with SNP + ODQ 83.5%), indicating that NO causes sympatholysis through a guanylyl cyclase signaling pathway. However, shear stress did not reduce constriction to PE (67.6 % vs. 68.1 %, and 67.6 %), indicating that increased shear stress during exercise is not the source of the NO causing sympatholysis. We conclude that nitric oxide acting through guanylyl cyclase causes sympatholysis, but the source of the nitric oxide during exercise is not shear stress-induced endothelial cell activation

The Effect of Shear Stress, Potassium, and Adenosine on α-1 Adrenergic Vasoconstriction of Rat Soleus Feed Arteries

During exercise, blood flow increases to the working skeletal muscle primarily because of dilation of the arteries and arterioles feeding the muscle. Sympathetic nerve activity also increases during exercise, augmenting the release of the neurotransmitter norepinephrine (NE) at the arterial wall and into the blood. NE acts to constrict blood vessels; however, arteries and arterioles within contracting skeletal muscle dilate despite the increased NE present. This has led to the concept of functional sympatholysis (4), the idea that a chemical released from contracting skeletal muscle interferes with NE signaling. NE acts by binding to adrenergic (alpha and beta) receptors, and it is alpha receptors in the arterial wall that cause vasoconstriction (8). While both α-1 and α-2 receptor subtypes have been found in some vascular beds of some species, there is significant evidence that in rat calf muscles, the response to norepinephrine is mediated solely by α-1 receptors (5, 9). Because α-1 receptors are the sole respondents to sympathetic signaling, we studied three proposed substances that may interfere with sympathetic signaling at the α-1 receptors, thereby mediating sympatholysis. There is evidence to suggest that heat and acidosis may partially mediate sympatholysis of α-1 receptors (1, 2). This study sought to determine whether increased levels of shear stress, potassium, or adenosine also contribute to sympatholysis. If shear stress, potassium, and adenosine are, in fact, sympatholytic agents, they will reduce the vasoconstriction mediated by the α-1 receptors in rat soleus muscle feed arteries. We hypothesized that all three variables would be sympatholytic agents

Effect of Shear Stress Direction on Endothelial Function and eNOS Phosphorylation in Soleus Feed Arteries

Blood flow feeding tissues and organs is closely regulated in order to meet metabolic and functional needs. Control of blood flow is accomplished by regulating the diameter of the arteries and arterioles feeding different organs. Several neural, hormonal, chemical and mechanical mechanisms contribute to the constriction and dilation of arteries. Shear stress, the frictional force created by streaming blood on the endothelial layer of arteries, is one of these mechanical mechanisms (1). Shear stress causes both acute and long term effects on endothelial cells (1,2,5). Blood in arteries typically flows away from the heart towards organs (causing antegrade shear stress) during cardiac contraction and briefly flows back toward the heart (causing retrograde shear stress) during cardiac filling. Retrograde flow occurs more often in some disease situations, and studies have shown that retrograde shear stress decreases endothelial cell function (3,4). The specific mechanisms for endothelial dysfunction are unknown, but altered mechanisms could include impaired cell signaling pathways. The most important endothelial cell dilatory signaling pathway is the production of nitric oxide (NO). Retrograde shear stress causes endothelial cells to secrete NO, and increased rates of shear stress cause increased expression and phosphorylation of nitric oxide synthase (eNOS). Regulatory phosphorylation of eNOS can potentially occur on at least four sites: Ser 1177, Ser 116, Ser 635 and Thr 497 (3). The most well characterized of these is Ser 1177, which is phosphorylated by a by PI3K/AKT shear dependent pathway. Regulating phosphorylation of eNOS is critical to endothelial health and maintaining cardiovascular equilibrium. Using rat soleus muscle feed arteries, we seek to determine the effects of changes in shear stress direction on both endothelial cell function and phosphorylation of eNOS at the Ser 1177 site