Transfer of nitric oxide by blood from upstream to downstream resistance vessels causes microvascular dilation.

The discovery that hemoglobin, albumin, and glutathione carry and release nitric oxide (NO) may have consequences for movement of NO by blood within microvessels. We hypothesize that NO in plasma or bound to proteins likely survives to downstream locations. To confirm this hypothesis, there must be a finite NO concentration ([NO]) in arteriolar blood, and upstream resistance vessels must be able to increase the vessel wall [NO] of downstream arterioles. Arteriolar blood NO was measured with NO-sensitive microelectrodes, and vessel wall [NO] was consistently 25-40% higher than blood [NO]. Localized suppression of NO production in large arterioles over 500-1,000 microm with L-nitroarginine reduced the [NO] approximately 40%, indicating as much as 60% of the wall NO was from blood transfer. Flow in mesenteric arteries was elevated by occlusion of adjacent arteries to induce a flow-mediated increase in arterial NO production. Both arterial wall and downstream arteriolar [NO] increased and the arterioles dilated as the blood [NO] was increased. To study receptor-mediated NO generation, bradykinin was locally applied to upstream large arterioles and NO measured there and in downstream arterioles. At both sites, [NO] increased and both sets of vessels dilated. When isoproterenol was applied to the upstream vessels, they dilated, but neither the [NO] or diameter downstream arterioles increased. These observations indicate that NO can move in blood from upstream to downstream resistance vessels. This mechanism allows larger vessels that generate large [NO] to influence vascular tone in downstream vessels in response to both flow and receptor stimuli.

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.

Obesity lowers hyperglycemic threshold for impaired in vivo endothelial nitric oxide function.

Obesity is a risk for type II diabetes mellitus and increased vascular resistance. Disturbances of nitric oxide (NO) physiology occur in both obese animals and humans. In obese Zucker rats, we determined whether a protein kinase C-beta II (PKC-beta II) mechanism may lower the resting NO concentration ([NO]) and predispose endothelial NO abnormalities at lower glucose concentrations than occur in lean rats. NO was measured with microelectrodes touching in vivo intestinal arterioles. At rest, the [NO] in obese Zucker rats was 60 nm less than normal or about a 15% decline. After local blockade of PKC-beta II with LY-333531, the [NO] increased approximately 90 nm in obese rats but did not change in lean rats. In lean rats, administration of 300 mg/dl D-glucose for 45 min depressed endothelium-dependent dilation; only 200 mg/dl was required in obese animals. These various observations indicate that resting [NO] is depressed in obese rats by a PKC-beta II mechanism and the hyperglycemic threshold for endothelial NO suppression is reduced to 200 mg/dl D-glucose.

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.

Integration of intestinal structure, function, and microvascular regulation.

Without an increase in blood flow to provide additional oxygen, intestinal absorption of nutrients cannot proceed. Studies of the intestinal microvascular structure and distribution of resistance indicated that most of the microvascular regulation must occur outside the mucosal tissues. This requires a communication system from the mucosa to resistance vessels unlike that of any other organ. The various mechanisms involved and their communication from mucosal to arteriolar cells has required an integrated study of intestinal structure, physiology, and microvascular regulation. The results of this analysis using diverse approaches have revealed some of the major physical and cellular mechanisms that couple intestinal absorption and microvascular function.

Mechanism of increased vessel wall nitric oxide concentrations during intestinal absorption.

Vasoactive compounds, including nitric oxide (NO) and hypertonic sodium, may diffuse from venous endothelial cells and blood to the arterial wall during intestinal absorption. This hypothesis was tested by measuring the perivascular NO concentration ([NO]) for paired small arteries and veins with NO-sensitive microelectrodes. Resting arterial and venous wall concentrations for nine vessel pairs (5 rats) were 353 +/- 28 and 401 +/- 48 (SE) nM. During mucosal absorption of 100 and 300 mg/dl glucose, the artery dilated 12 +/- 1.5 and 17 +/- 2%, [NO] increased to 540 +/- 68 and 550 +/- 49 nM, and venous wall [NO] increased to 557 +/- 60 and 633 +/- 70 nM. During venous occlusion to block diffusion of materials from venous blood to the artery wall, the arterial and venous [NO] decreased by 70-80%, and one-half of the arterial dilation subsided. Superfusion with 320 and 360 mosmol/l hypertonic sodium medium to simulate the sodium hyperosmolarity during mucosal absorption of glucose increased the arterial [NO] by 20-30 and 40-50%; 360 mosmol/l saline made hypertonic with mannitol did not significantly increase the [NO]. Although venous to arterial diffusion of NO occurred, the increased arterial [NO] during mucosal glucose absorption was primarily generated by the arterial wall in response to materials that diffused from venous blood, such as hypertonic sodium.

Non-insulin-dependent diabetes and hyperglycemia impair rat intestinal flow-mediated regulation.

Release of nitric oxide from small arteries and larger arterioles of the intestine maintains their dilation and thereby supports mucosal blood flow. This flow-dependent mechanism can be studied by isosmotic replacement of sodium chloride with mannitol over the mucosa to lower mucosal metabolism and blood flow requirements. We tested the hypothesis that flow-mediated regulation is impaired in the non-insulin-dependent Zucker fatty diabetic (ZFD) male rats because of their marginally impaired endothelium-dependent dilation. Furthermore, we determined whether the depressed acetylcholine dilation associated with acute hyperglycemia in normoglycemic Zucker (NZ) rats also impairs flow-mediated regulation. When mannitol replaced sodium chloride over the villi, intestinal blood flow decreased significantly (P < 0.05) less in ZFD (80.9 +/- 6.8% of control) than NZ rats (40.9 +/- 6.4% of control). After 300 mg/dl hyperglycemia for 30 min, normal arterioles had impaired responses to acetylcholine and the resting blood flow and oxygen consumption were suppressed about 60%, which indicate the importance of basal nitric oxide release for intestinal vascular support of metabolism. The evidence of impaired flow-mediated dilation in ZFD and decreased resting blood flow after hyperglycemia in NZ rats demonstrated that both acute and chronic hyperglycemia disturb endothelial regulation of the intestinal vasculature.

Time- and order-dependent changes in functional and NO-mediated dilation during exercise training.

Arterial vessel responses to sodium nitroprusside (SNP) and acetylcholine (ACh) were measured in the spinotrapezius muscle of sedentary (Sed) and treadmill-trained (Tr) rats to determine whether these endothelium-dependent (ACh) and -independent (SNP) mechanisms contribute to the training-induced increase in functional vasodilation previously observed. Control and maximal vessel diameters were similar between Sed and Tr. After 8 wk of training, functional dilation (2-, 4-, and 8-Hz contractions) was enhanced in all orders of vessels studied [terminal feed artery (FA), largest arterioles (1A), and intermediate-sized arterioles (2A)], but responses to SNP were increased only in FA. Responses to ACh were not significantly increased in any vessel order. After 16 wk of training, functional dilation had regressed in Tr such that only the FA response to 4 Hz was significantly elevated relative to Sed. However, the FA and 1A responses to SNP were significantly greater in Tr than in Sed, as were the 1A and 2A responses to ACh. These results show a dissociation of functional dilation and SNP- or ACh-mediated responses, as well as age-dependent interactions, a time-dependent progression, and vessel order specificity in the adaptations to training.

Aldose reductase and IGF-I gene expression in aortic and arteriolar smooth muscle during hypo- and hyperinsulinemic diabetes.

Two genes whose expression is likely to be altered during diabetes mellitus are aldose reductase (AD) and insulin-like growth factor-I (IGF-I). We proposed that gene expression of AD is increased in vascular smooth muscle during diabetes mellitus due to hyperglycemia, while IGF-I expression is decreased in insulin-deficient diabetes and elevated in insulin-resistant diabetes. The mRNA for both was measured in the renal glomerulus, in the vascular smooth muscle of large arterioles from the brain, kidney, and small intestine, and in the aorta of hypoinsulinemic streptozotocin (STZ)-treated rats and hyperinsulinemic Zucker diabetic fatty (ZDF) rats. Quantitative in situ hybridization was used to determine variations in expression. Expression of the AD gene was unchanged in STZ and ZDF rats, except for a decrease of about 50% in glomeruli and renal smooth muscle of STZ diabetic rats. Expression of IGF-I generally decreased in vascular smooth muscle of insulin-depleted STZ diabetic rats, but was normal in hyperinsulinemic ZDF rats. The data indicate that decreased expression of the AD gene is a specific problem in renal vascular smooth muscle and glomeruli in the insulin-depleted STZ model of diabetes. The expression of the IGF-I gene in vascular muscle was decreased in hypoinsulinemic diabetic animals, but did not increase in hyperinsulinemic diabetic rats.

Intestinal absorption of sodium and nitric oxide-dependent vasodilation interact to dominate resting vascular resistance.

The villi of the small intestine maintain a hypertonic interstitium at all times, and the submucosal glands constantly secrete ions and accompanying water into the lumen. Generation of the 400- to 600-mOsm interstitial fluid in the villus and secretion by glands may require a large expenditure of energy and, consequently, have major effects on intestinal vascular regulation to supply oxygen and nutrients. Blood flow and oxygen consumption were measured in the ileum of anesthetized rats during natural resting conditions with physiological sodium chloride in the bathing fluid and during isosmotic replacement of sodium chloride with mannitol. Microvascular pressures and blood flow were used to determine the changes in resistance of the major arterioles and the terminal vasculature. When mannitol replaced sodium chloride in contact with the villi, intestinal blood flow decreased to 58.6 +/- 2.8% of control, and oxygen consumption was 54.2 +/- 3.4% of control. Resistance of the major arterioles increased 101.7 +/- 9.9%, and that of the terminal vasculature increased 40.4 +/- 6.2%. The increased resistance appeared to be caused by suppression of a nitric oxide mechanism. Local application of 10(-4) mol/L NG-nitro-L-arginine methyl ester caused about the same reduction in flow and increases in regional vascular resistance as during replacement of sodium but did not alter the oxygen consumption. These data indicate that about half of the intestinal metabolic rate during natural resting conditions is devoted to sodium secretion/absorption. Large resistance vessels are dilated to maintain a high blood flow through release of nitric oxide. We propose that dilation of the terminal vasculature in the metabolically active tissues increased flow velocity sufficiently in the major resistance vessels to cause a flow-mediated release of nitric oxide.

Resting oxygenation of rat and rabbit intestine: arteriolar and capillary contributions.

Counter-current exchange of oxygen may occur between inflow and outflow microvessels of the small intestine and greatly influence the dominant sites of tissue oxygenation. To determine the location and magnitude of potential exchange, percent saturation of hemoglobin with oxygen (%SHb) was measured in microvessels throughout the intestine of rats and rabbits. Oxygen losses from systemic arterial blood through large and intermediate arterioles (second order, 2A) was 5-7%SHb in both species, and there was no evidence of an increase in percent saturation along intermediate and large venules. A larger loss of oxygen from arterioles and an increase in venous saturation would be evident if significant arteriolar to venular counter-current exchange of oxygen occurred in the submucosa. From 2A to the villus tip, arteriolar saturation decreased approximately 10%SHb in rabbits and approximately 15%SHb in rats; the villus tip percent saturation was 72.9 +/- 3.9%SHb in rabbits and 69.9 +/- 2.9%SHb in rats. An additional decrease of 5%SHb in rabbits and 15%SHb in rats occurred across the villus capillaries and smallest venules. Although the total reduction in percent saturation across the villi was different between the two species, 70-90% of the total arteriovenous oxygen losses occurred in the capillaries and small arterioles of the villi. We found no evidence of counter-current exchange of oxygen in villi or any other vascular region. Rather, as appears to occur in most organs, small arterioles in conjunction with capillaries dominate resting oxygen exchange to tissue.

Endothelial-dependent vasodilation is preserved in non-insulin-dependent Zucker fatty diabetic rats.

Alterations in the structural properties of the microvasculature and in vasodilation mediated by endothelial- and, to some extent, nonendothelial-dependent mechanisms occurs in insulin-dependent diabetic humans and animals. Less severe problems of this type appear to occur during non-insulin-dependent diabetes mellitus (NIDDM) in humans, but data based on animal models of NIDDM are not available. The endothelial- and nonendothelial-mediated dilation of intestinal arterioles was studied in insulin-resistant male Zucker fatty diabetic (DB) rats and their lean normal male littermates (LM) at ages 22-25 and 35-40 wk. DB become hyperglycemic (450-550 mg/100 ml) at age 9-10 wk. Microiontophoretic release of acetylcholine, ADP, and nitroprusside onto arterioles caused equivalent dilation in LM and DB for both large and intermediate diameter arterioles. Administration of streptozotocin (STZ) to DB at age 18-19 wk lowered their insulin concentration approximately 25% but did not significantly effect the resting plasma glucose concentration. However, endothelial-dependent vasodilation was attenuated by 70-80% within 8-10 wk. The overall results indicate that prolonged hyperglycemia in insulin-resistant but hyperinsulinemic rats does not impair the endothelial- and nonendothelial-dependent dilation of the intestinal microvasculature. However, compromising beta-cell function with STZ, as indicated by lowering the insulin concentration by one-fourth, substantially compromises endothelial-dependent dilation similar to that found in insulin-dependent diabetic rats and humans.

Vascular endothelium and smooth muscle remodeling accompanies hypertrophy of intestinal arterioles in streptozotocin diabetic rats.

The purpose of this study was to document alterations in endothelial and smooth muscle cell morphology of first- and second-order intestinal arterioles after 6 months of streptozotocin-induced diabetes. Both light and scanning electron microscopic techniques were used to quantitate the changes in the microvasculature. After rendering the first- and second-order intestinal arterioles passive and processing the vessels, it was determined that these microvessels were significantly dilated in the diabetic animals. Further examination revealed that in the diabetic animals, the cross-sectional area of the endothelial layer was increased in both 1A and 2A vessels, and the smooth muscle layer cross-sectional area was significantly increased in 1A vessels. Individual smooth muscle cells were significantly increased in width in the diabetic animals, but not in length. These data suggest that in this model of diabetes in rats, intestinal arteriolar hypertrophy was accompanied by significant remodeling of the arteriolar wall.

Excess oxygen delivery during muscle contractions in spontaneously hypertensive rats.

These experiments determined whether a deficit in oxygen supply relative to demand could account for the sustained decrease in tissue PO2 observed during contractions of the spinotrapezius muscle in spontaneously hypertensive rats (SHR). Relative changes in blood flow were determined from measurements of vessel diameter and red blood cell velocity. Venular hemoglobin oxygen saturation measurements were performed by using in vivo spectrophotometric techniques. The relative dilation [times control (xCT)] of arteriolar vessels during contractions was as large or greater in SHR than in normotensive rats (Wistar-Kyoto), as were the increases in blood flow (2 Hz, 3.50 +/- 0.69 vs. 3.00 +/- 1.05 xCT; 4 Hz, 10.20 +/- 3.06 vs. 9.00 +/- 1.48 xCT; 8 Hz, 16.40 +/- 3.95 vs. 10.70 +/- 2.48 xCT). Venular hemoglobin oxygen saturation was lower in the resting muscle of SHR than of Wistar-Kyoto rats (31.0 +/= 3.0 vs. 43.0 +/- 1.9%) but was higher in SHR after 4- and 8-Hz contractions (4 Hz, 52.0 +/- 4.8 vs. 43.0 +/- 3.6%; 8 Hz, 51.0 +/- 4.6 vs. 41.0 +/- 3.6%). Therefore, an excess in oxygen delivery occurs relative to oxygen use during muscle contractions in SHR. The previous and current results can be reconciled by considering the possibility that oxygen exchange is limited in SHR by a decrease in anatomic or perfused capillary density, arteriovenular shunting of blood, or decreased transit time of red blood cells through exchange vessels.

Role of a lymphatic system in glucose absorption and the accompanying microvascular hyperemia.

In this study we evaluated the importance of a functional intestinal lymphatic system on changes in arteriolar and venular blood oxygen content, vasodilation, and elevation of venous blood osmolarity during glucose absorption. Glucose absorption was associated with a doubling of the arteriovenous oxygen difference [(A-V)O2], a 50 mosM increase in venous blood osmolarity, and 17% dilation of the intermediate-diameter arterioles. After the lymph vessels were mechanically blocked with mineral oil, glucose absorption again doubled the (A-V)O2, indicating that glucose was absorbed without a functional lymphatic system. Furthermore, venous blood osmolarity and arteriolar diameter increased similarly with and without a functional lymphatic system. This study indicates that even though the lymphatic system likely facilitates distribution of hypertonic material in the bowel wall during absorption, blockade of the lymphatics did not appreciably hinder vasodilation, glucose absorption, changes in intravascular oxygen content, or the elevation of tissue hyperosmolarity, as judged by the tonicity of the venular blood. Therefore, passage of materials absorbed or released in the mucosa to the submucosa through venular blood flow may be very important to the mechanism of absorptive hyperemia.

Active and passive arteriolar regulation in spontaneously hypertensive rats.

This study determined to what extent active and passive wall tensions increase in in vivo intestinal arterioles of 13- to 15-week-old and 25- to 27-week-old spontaneously hypertensive rats (SHR) to maintain normal or smaller arteriolar diameters during microvascular hypertension. Acetylcholine and nitroprusside were used to determine whether vascular muscle relaxation to endothelium-derived relaxing factor or cyclic GMP is impaired. Large arterioles of hypertensive rats have passive tension-circumference relations that are steeper and shifted to the left compared with those of age-matched controls; passive resistance to distension limits vasodilation in hypertensive rats except at their naturally elevated arteriolar pressure. Passive tension contributes approximately 30% of the total resting tension in arterioles of hypertensive and normotensive rats because a greater passive tension occurs at the 20% to 25% constricted resting diameter in hypertensive rats. Absolute and relative changes in the diameter of SHR arterioles during acetylcholine and nitroprusside application were equal to or greater than those in Wistar-Kyoto rats. However, reduction in active tension was suppressed in older SHR and remained approximately 50% higher than that found in older Wistar-Kyoto rats during drug application. Vasoconstriction and increased passive resistance to distension of the arteriolar wall diminish the active tension required to maintain normal or smaller resting diameters against microvascular hypertension. However, the elevated microvascular pressure in hypertensive rats is required to allow near-normal dilation to compensate for their increased passive resistance to stretch and decreased ability to relax active tension through cyclic GMP mechanisms.

Adrenergic and pressure-dependent vascular regulation in sedentary and trained rats.

In this study, we determined if aerobic exercise training alters adrenergic or pressure-dependent vascular regulation in the rat hindlimb or intestine. Pressor responses to bilateral carotid artery occlusion and systemic phenylephrine (PE) infusion were not altered by training. During occlusion, peak and steady-state changes in hindlimb vascular resistance (HLR) were significantly greater in trained (24 and 13%) than in sedentary (8 and -3%) rats; a similar trend existed for intestinal vascular resistance (IR). The pressure-dependent contribution was consistent between groups (HLR: peak 55-85%, steady state 25-45%; IR: peak and steady state 40-65%). During PE infusion, increases in IR and HLR were similar between groups. The increase in HLR was substantially pressure dependent in both groups (approximately 50% at highest dose) as was the change in IR in trained rats. However, the IR response to PE was not pressure dependent in sedentary rats. The direct effects of PE were similar between sedentary and trained rats in the hindlimb but were suppressed in the intestine of trained rats compared with sedentary rats. Therefore, aerobic exercise training altered adrenergic and pressure-dependent vasoregulatory mechanisms in both skeletal muscle and intestinal tissues.