Reduced perivascular PO2 increases nitric oxide release from endothelial cells.

Many studies have suggested that endothelial cells can act as "oxygen sensors" to large reductions in oxygen availability by increasing nitric oxide (NO) production. This study determined whether small reductions in oxygen availability enhanced NO production from in vivo intestinal arterioles, venules, and parenchymal cells. In vivo measurements of perivascular NO concentration ([NO]) were made with NO-sensitive microelectrodes during normoxic and reduced oxygen availability. During normoxia, intestinal first-order arteriolar [NO] was 397 +/- 26 nM (n = 5), paired venular [NO] was 298 +/- 34 nM (n = 5), and parenchymal cell [NO] was 138 +/- 36 nM (n = 3). During reduced oxygen availability, arteriolar and venular [NO] significantly increased to 695 +/- 79 nM (n = 5) and 534 +/- 66 nM (n = 5), respectively, whereas parenchymal [NO] remained unchanged at 144 +/- 34 nM (n = 4). During reduced oxygenation, arteriolar and venular diameters increased by 15 +/- 3% and 14 +/- 5%, respectively: NG-nitro-L-arginine methyl ester strongly suppressed the dilation to lower periarteriolar Po2. Micropipette injection of a CO2 embolus into arterioles significantly attenuated arteriolar dilation and suppressed NO release in response to reduced oxygen availability. These results indicated that in rat intestine, reduced oxygen availability increased both arteriolar and venular NO and that the main site of NO release under these conditions was from endothelial cells.

Arteriolar nitric oxide concentration is decreased during hyperglycemia-induced betaII PKC activation.

betaII protein kinase C (betaPKC) is activated during acute and chronic hyperglycemia and may alter endothelial cell function. We determined whether blockade of betaPKC protected in vivo endothelial formation of NO, as measured with NO-sensitive microelectrodes in the rat intestinal vasculature. NaCl hyperosmolarity, a specific endothelial stimulus to increase NO formation, caused approximately 20% arteriolar vasodilation and approximately 30% increase in NO concentration ([NO]). After topical 300 mg/dl hyperglycemia for 45 min, both responses were all but abolished. In comparison, pretreatment with LY-333531, a specific betaPKC inhibitor, maintained vasodilation and [NO] responses to NaCl hyperosmolarity after hyperglycemia. The betaPKC inhibitor alone had no significant effects on resting diameter or [NO] or their responses to NaCl hyperosmolarity. In separate rats, after topical hyperglycemia had suppressed dilation to ACh, LY-333531 restored approximately 70% of the dilatory response. These data demonstrated that activation of betaPKC during acute hyperglycemia depressed in vivo endothelial formation of NO at rest and during stimulation. This abnormality can be minimized by inhibition of betaPKC before hyperglycemia and can be substantially reversed by PKC inhibition after hyperglycemia-induced abnormalities have occurred.

Dependence of intestinal arteriolar regulation on flow-mediated nitric oxide formation.

Our hypothesis was that a large fraction of resting nitric oxide (NO) formation is driven by flow-mediated mechanisms in the intestinal microvasculature of the rat. NO-sensitive microelectrodes measured the in vivo perivascular NO concentration ([NO]). Flow was increased by forcing the arterioles to perfuse additional nearby arterioles; flow was decreased by lowering the mucosal metabolic rate by reducing sodium absorption. Resting periarteriolar [NO] of large arterioles (first order; 1A) and intermediate-sized arterioles (second order; 2A) was 337 +/- 20 and 318 +/- 21 nM. The resting [NO] was higher than the dissociation constant for the NO-guanylate cyclase reaction of vascular smooth muscle; therefore, resting [NO] should be a potent dilatory signal at rest. Over flow velocity and shear rate ranges of approximately 40-180% of control, periarteriolar [NO] changed 5-8% for each 10% change in flow velocity and shear rate. The relationship of [NO] to flow velocity and shear rate demonstrated that 60-80% of resting [NO] depended on flow-mediated mechanisms. Therefore, moment-to-moment regulation of [NO] at rest is an ongoing process that is highly dependent on flow-dependent mechanisms.

Acute hyperglycemia depresses arteriolar NO formation in skeletal muscle.

In the rat intestinal and cerebral microvasculatures, acute D-glucose hyperglycemia suppresses endothelium-dependent dilation to ACh without affecting endothelium-independent dilation to nitroprusside. This study determined whether acute hyperglycemia suppressed arteriolar wall nitric oxide concentration ([NO]) at rest or during ACh stimulation and inhibited nitroprusside-, ACh- or contraction-induced dilation of rat spinotrapezius arterioles. Vascular responses were measured before and after 1 h of topical 300 mg/100 ml D-glucose; arteriolar [NO] was measured with NO-sensitive microelectrodes. Arteriolar dilation to ACh was not significantly altered after superfusion of 300 mg/100 ml D-glucose. However, after hyperglycemia, arteriolar [NO] was not increased by ACh, compared with a 300 nM increase attained during normoglycemia. Arteriolar dilation to submaximal nitroprusside and muscle contractions was enhanced by hyperglycemia. These results indicated that in the rat spinotrapezius muscle, acute hyperglycemia suppressed arteriolar NO production while simultaneously augmenting vascular smooth muscle responsiveness to nitroprusside, presumably through cGMP-mediated mechanisms. In effect, this may have allowed ACh- and muscle contraction-induced vasodilation to be maintained during hyperglycemia despite an impaired NO system.

Postjunctional alpha 2-adrenoceptors are not present in proximal arterioles of rat intestine.

The purpose of this study was to evaluate two potential stimuli for nitric oxide (NO) release in rat intestinal arterioles during sympathetic nerve activation. To determine whether these vessels contain endothelial alpha 2-adrenoceptors linked to the L-arginine-NO pathway, intravital microscopy was used to study the response of first-order arterioles (1As, 20-40 microns ID) to direct application of 1) the selective alpha 2-agonist BHT-933 and 2) norepinephrine (NE) or sympathetic nerve stimulation before and after alpha 1- or alpha 2-receptor blockade. The effect of sympathetic nerve stimulation on 1A wall shear rate (WSR) was also determined to evaluate the possibility of hemodynamic shear stress as a stimulus for NO release. BHT-933 had no effect on 1A diameter, whereas NE produced dose-dependent constrictions of 5 +/- 3 to 15 +/- 3 microns, which were usually abolished by the alpha 1-antagonist prazosin but unaffected by the alpha 2-antagonist idazoxan. Sympathetic nerve stimulation at 3, 8, and 16 Hz induced constrictions of 4 +/- 1, 8 +/- 2, and 17 +/- 4 microns, respectively, and these constrictions were also usually abolished by prazosin but unaffected by idazoxan. Resting WSR averaged 1,997 +/- 163 s-1 and decreased to 1,587 +/- 209, 1,087 +/- 195, and 537 +/- 99 s-1 during 3-, 8-, and 16-Hz nerve stimulation. These results suggest that alpha 2-adrenoceptor-dependent pathways do not influence either resting tone or sympathetic constriction of proximal arterioles in the intestinal submucosa and that luminal shear stress in these vessels significantly decreases with sympathetic constriction. It therefore appears unlikely that either alpha 2-receptor activation or changes in hemodynamic shear serve as stimuli for arteriolar NO release during periods of increased sympathetic nerve activity.

Endothelium-derived nitric oxide limits sympathetic neurogenic constriction in intestinal microcirculation.

We have recently shown that endogenous nitric oxide (NO) activity can attenuate the sympathetic neurogenic constriction of intestinal arterioles. The purpose of this study was to determine whether the microvascular endothelium is an important site of NO production under these conditions. In the superfused small intestine of the rat, intravital microscopy was used to study the responses of first-order arterioles (1A) to perivascular sympathetic nerve stimulation and directly applied norepinephrine before and then after passage of a CO2 embolus through the 1A lumen to inhibit endothelial function. CO2 embolization did not significantly alter resting arteriolar diameter (50 +/- 4 microns before vs. 51 +/- 4 microns after embolization) but abolished the dilator response to acetylcholine without altering the dilator response to sodium nitroprusside. Stimulation at 3, 8, and 16 Hz caused respective constrictions of 4 +/- 1, 11 +/- 1, and 18 +/- 2 microns, and after CO2 these responses were significantly increased to 9 +/- 1, 18 +/- 1, and 29 +/- 3 microns, respectively. Exposure to the nitric oxide synthase inhibitor NG-monomethyl-L-arginine(10(-4) M in superfusate) after CO2 embolization had no further effect on the magnitude of neurogenic constriction. Similar results were seen when embolization was achieved with N2, and CO2 embolization had the same effect on norepinephrine-induced constriction as it did on neurogenic constriction. These results suggest that nitric oxide of endothelial origin can attenuate sympathetic neurogenic constriction in the intestinal microvasculature.

Modulation of sympathetic constriction by the arteriolar endothelium does not involve the cyclooxygenase pathway.

We have recently shown that the responsiveness of rat intestinal arterioles to increased sympathetic nerve activity is modulated by the actions of endothelial-derived nitric oxide. Because the microvascular endothelium can also produce vasodilator prostaglandins, the purpose of this study was to determine if endogenous cyclooxygenase products also limit sympathetic arteriolar constriction in this vascular bed. Intravital microscopy was used to study the responses of small feed arteries, first-order arterioles and second-order arterioles to perivascular sympathetic nerve stimulation in the superfused rat small intestine. Stimulation at 3, 8 and 16 Hz caused frequency-dependent constrictions of each vessel type that are abolished by the alpha-adrenoceptor antagonist phentolamine (10(-6) M superfusate concentration). The cyclooxygenase inhibitor meclofenamate (3 x 10(-5) M superfusate concentration) completely abolished the dilator responses to topically applied arachidonic acid, but had no effect on the magnitude or rate of sympathetic constriction in any vessel type. These results suggest that endogenous cyclooxygenase activity does not influence sympathetic tone in the intestinal microvasculature.

Nitric oxide modulates arteriolar responses to increased sympathetic nerve activity.

The purpose of this study was to determine whether arteriolar responses to increased sympathetic nerve activity are limited by the actions of endogenous nitric oxide. Intravital microscopy was used to examine diameter responses of small feed arteries (SFA), first-order arterioles (1A) and second-order arterioles (2A) to perivascular sympathetic nerve stimulation in the superfused rat small intestine. Stimulation induced a frequency-dependent constriction in all vessel types that was completely abolished by the alpha-adrenoceptor antagonist phentolamine (10(-6) M). In SFA and 1A, the magnitude of sympathetic constriction was increased significantly in the presence of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine(L-NMMA, 10(-4) M). In SFA (n = 7), stimulation at 3, 8, and 16 Hz induced constrictions of 11 +/- 1, 28 +/- 4, and 42 +/- 3%, respectively, under the normal superfusate vs. 28 +/- 3, 46 +/- 5, and 76 +/- 3% in the presence of L-NMMA. For 1A (n = 7), stimulation induced constrictions of 10 +/- 1, 27 +/- 4, and 37 +/- 3% under the normal superfusate vs. 24 +/- 2, 47 +/- 3, and 72 +/- 4% in the presence of L-NMMA. The effect of L-NMMA on sympathetic constriction in SFA (n = 7) was completely reversed by the additional presence of 5 x 10(-3) M L-arginine in the superfusate. These results suggest that endogenous nitric oxide activity can attenuate sympathetic neurogenic constriction in the intestinal microvasculature.