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Myogenic vasoconstriction, an intrinsic response to elevated transmural pressure (TMP), requires the activation of sphingosine kinase (Sk1) and the generation of reactive oxygen species (ROS). We hypothesized that pressure-induced Sk1 signaling and ROS generation are functionally linked. Using a model of cannulated resistance arteries isolated from the hamster gracilis muscle, we monitored vessel diameter and smooth muscle cell (SMC) Ca2+i (Fura-2) or ROS production (dichlorodihydrofluorescein). Elevation of TMP stimulated the translocation of a GFP-tagged Sk1 fusion protein from the cytosol to the plasma membrane, indicative of enzymatic activation. Concurrently, elevation of TMP initiated a rapid and transient production of ROS, which was enhanced by expression of wild-type Sk1 (hSk(wt)) and inhibited by its dominant-negative mutant (hSk(G82D)). Exogenous sphingosine-1-phosphate (S1P) also stimulated ROS generation is isolated vessels. Chemical (1 micromol/L DPI), peptide (gp91ds-tat/gp91ds), and genetic (N17Rac) inhibition strategies indicated that NADPH oxidase was the source of the pressure-induced ROS. NADPH oxidase inhibition attenuated myogenic vasoconstriction and reduced the apparent Ca2+ sensitivity of the SMC contractile apparatus, without affecting Ca2+-independent, RhoA-mediated vasoconstriction in response to exogenous S1P. Our results indicate a mandatory role for Sk1/S1P in mediating pressure-induced, NADPH oxidase-derived ROS formation. In turn, ROS generation appears to increase Ca2+ sensitivity, necessary for full myogenic vasoconstriction.

Objective The mechanisms regulating spiral modiolar artery (SMA) tone are not known, yet their characterization is pivotal for understanding inner ear blood flow regulation. Sphingosine-1-phosphate (S1P), known to stimulate vasoconstriction in several vascular beds, is a candidate regulator of SMA tone with potential pathophysiological relevance. Methods Gerbil SMAs were isolated, cannulated and pressurized (30 mm Hg transmural) for experimentation under near-in vivo conditions. For functional experiments, vascular diameter and intracellular Ca2+ were simultaneously measured. Standard RT-PCR and immunohistochemical techniques were also employed. Results mRNA transcripts encoding sphingosine kinase, S1P phosphohydrolase and three S1P receptors (S1P1–3) were detected in the SMA. S1P induced dose-dependent vasoconstriction of the SMA (EC50=115nmol/L), and enhanced the apparent Ca2+-sensitivity of the contractile apparatus. Noradrenaline did not elicit vasoconstriction. The Rho kinase inhibitor Y27632 (1μmol/L) reversed S1P-induced vasoconstriction and the S1P-mediated enhancement of Ca2+-sensitivity. RhoA was observed to translocate to the plasma membrane in response to stimulation with 30μmol/L S1P. Conclusion We conclude that all key signalling pathway constituents are present at the mRNA level for S1P to act as an endogenous regulator of SMA tone. S1P stimulates potent, RhoA/Rho kinase-dependent SMA vasoconstriction and Ca2+ sensitization. The high sensitivity to S1P suggests that SMA vasoconstriction is likely to occur under pathological conditions that increase intramural S1P concentrations (i.e., inflammation). From a clinical perspective, the present study identifies new potential therapeutic targets for the treatment of vascular-based, “stroke-like” inner ear pathologies: the enzymes responsible for S1P bioavailability and the S1P receptors.

Ovarian granulosa cells are coupled via gap junctions containing connexin43 (Cx43 or α-1 connexin). In the absence of Cx43, granulosa cells stop growing in an early preantral stage. However, the fact that granulosa cells of mature follicles express multiple connexins complicated interpretation of this finding. The present experiments were designed to clarify the role of Cx43 vs. these other connexins in the earliest stages of folliculogenesis. Dye injection experiments revealed that granulosa cells from Cx43 knockout follicles are not coupled, and this was confirmed by ionic current injections. Furthermore, electron microscopy revealed that gap junctions are extremely rare in mutant granulosa cells. In contrast, mutant granulosa cells were able to form gap junctions with wild-type granulosa cells in a dye preloading assay. It was concluded that mutant granulosa cells contain a population of connexons, composed of an unidentified connexin, that do not normally contribute to gap junctions. Therefore, although Cx43 is not the only gap junction protein present in granulosa cells of early preantral follicles, it is the only one that makes a significant contribution to intercellular coupling.

To characterize the nonuniform diameter response in a blood vessel after a given stimulus (e.g., arteriolar conducted response), frequent serial diameter measurements along the vessel length are required. We used an advanced image analysis algorithm (the "discrete dynamic contour") to develop a quick, reliable method for serial luminal diameter measurements along the arteriole visualized by intravital video microscopy. With the use of digitized images of the arteriole and computer graphics, the method required an operator to mark the image of the two inner edges of the arteriole at several places along the arteriolar length. The algorithm then "filled in" these marks to generate two continuous contours that "hugged" these edges. A computer routine used these contours to determine luminal diameters every 20 microm. Based on these diameters and on Poiseuille's law, the routine also estimated the hemodynamic resistance of the blood vessel. To demonstrate the usefulness of the method, we examined the character of spatial decay of KCl-induced conducted constriction along approximately 500-microm-long arteriolar segments and the KCl-induced increase in hemodynamic resistance computed for these segments. The decay was only modestly fitted by a simple exponential, and the computed increase in resistance (i.e., 5- to 70-fold) was only modestly predicted by resistance increase based on our mathematical model involving measurements at two arteriolar sites (Tyml K, Wang X, Lidington D, and Oullette Y. Am J Physiol Heart Circ Physiol 281: H1397-H1406, 2001). We conclude that our method provides quick, reliable serial diameter measurements. Because the change in hemodynamic resistance could serve as a sensitive index of conducted response, use of this index in studies of conducted response may lead to new mechanistic insights on the response.

Our recent in vitro study (Lidington et al. J Cell Physiol 185: 117–125, 2000) suggested that lipopolysaccharide (LPS) reduces communication along blood vessels. The present investigation extended this study to determine whether any effect of LPS and/or inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1β, and IL-6] on endothelial cell coupling in vitro could also be demonstrated for an arteriolar conducted response in vivo. Using an electrophysiological approach in monolayers of microvascular endothelial cells, we found that LPS (10 μg/ml) but not these cytokines reduced intercellular conductance ( c i) (an index of cell communication) and that LPS together with these cytokines did not further reduce c i. Also, c i was restored after LPS washout, and the LPS-induced reduction was prevented by protein tyrosine kinase (PTK) inhibitors (1.5 μM Tyr A9 and 10 nM PP-2). In our in vivo experiments in arterioles of the mouse cremaster muscle, local electrical stimulation evoked vasoconstriction that conducted along arterioles. LPS in the muscle superfusate did not alter local vasoconstriction but reduced the conducted response. Washout of LPS restored the conducted response, whereas PTK inhibitors prevented the effect of LPS. On the basis of a newly developed mathematical model, the LPS-induced reduction in conducted response was predicted to reduce the arteriolar ability to increase resistance to blood flow. We conclude that LPS can reduce communication in in vitro and in vivo systems comparably in a reversible and tyrosine kinase-dependent manner. Based on literature and present results, we suggest that LPS may compromise microvascular hemodynamics at both the arteriolar responsiveness and the conduction levels.

Septic patients have low plasma ascorbate concentrations and compromised microvascular perfusion. The purpose of the present experiments was to determine whether ascorbate improves capillary function in volume-resuscitated sepsis. Cecal ligation and perforation (CLP) was performed on male Sprague-Dawley rats. The concentration of ascorbate in plasma and urine, mean arterial blood pressure, and density of continuously perfused capillaries in the extensor digitorum longus muscle were measured 24 h after surgery. CLP caused a 50% decrease (from 56 +/- 4 to 29 +/- 2 microM) in plasma ascorbate concentration, 1,000% increase (from 46 +/- 13 to 450 +/- 93 microM) in urine ascorbate concentration, 20% decrease (from 115 +/- 2 to 91 +/- 2 mmHg) in mean arterial pressure, and 30% decrease (from 24 +/- 1 to 17 +/- 1 capillaries/mm) in the density of perfused capillaries, compared with time-matched controls. A bolus of intravenous ascorbate (7.6 mg/100 g body wt) administered immediately after the CLP procedure increased plasma ascorbate concentration and restored both blood pressure and density of perfused capillaries to control levels. In vitro experiments showed that ascorbate (100 microM) inhibited replication of bacteria and prevented hydrogen peroxide injury to cultured microvascular endothelial cells. These results indicate that ascorbate is lost in the urine during sepsis and that a bolus of ascorbate can prevent microvascular dysfunction in the skeletal muscle of septic animals. Our study supports the view that ascorbate may be beneficial for patients with septic syndrome.