Microvascular dilation in response to occlusion: a coordinating role for conducted vasomotor responses

In rat cremasteric microcirculation, mechanical occlusion of one branch of an arteriolar bifurcation causes an increase in flow and vasodilation of the unoccluded daughter branch. This dilation has been attributed to the operation of a shear stress-dependent mechanism in the microcirculation. Instead of or in addition to this, we hypothesized that the dilation observed during occlusion is the result of a conducted signal originating distal to the occlusion. To test this hypothesis, we blocked the ascending spread of conducted vasomotor responses by damaging the smooth muscle and endothelial cells in a 200-microm segment of second- or third-order arterioles. We found that a conduction blockade eliminated or diminished the occlusion-associated increase in flow through the unoccluded branch and abolished or strongly attenuated the vasodilatory response in both vessels at the branch. We also noted that vasodilations induced by ACh (10(-4) M, 0.6 s) spread to, but not beyond, the area of damage. Taken together, these data provide strong evidence that conducted vasomotor responses have an important role in coordinating blood flow in response to an arteriolar occlusion.     

Brimonidine evokes heterogeneous vasomotor response of retinal arterioles: diminished nitric oxide-mediated vasodilation when size goes small

Brimonidine, an alpha2-adrenergic receptor (AR) agonist, has been employed in the treatment of glaucoma due to its beneficial effects on intraocular pressure reduction and neuroprotection. In addition, some studies have implicated that brimonidine might influence ocular blood flow; however, its effect on the retinal microcirculation has not been documented. Herein, we examined the vasomotor action of brimonidine on different branching orders of retinal arterioles in vitro and determined the contribution of the alpha2-AR subtype and the role of endothelium-derived nitric oxide (NO) in this vasomotor response. First- and second-order retinal arterioles of pigs were isolated, cannulated, and pressurized for functional studies. Videomicroscopic techniques were employed to record diameter changes in response to brimonidine. RT-PCR was performed for detection of alpha-AR and endothelial NO synthase (eNOS) mRNA in retinal arterioles. All first-order arterioles (82 +/- 2 microm ID) dilated dose dependently to brimonidine (0.1 nM to 10 microM) with 10% dilation at the highest concentration. Second-order arterioles (50 +/- 1 microm ID) responded heterogeneously with either dilation or constriction. The incidence and magnitude of vasoconstriction were increased with increasing brimonidine concentration. Administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester abolished the brimonidine-induced vasodilation in first- and second-order arterioles. Regardless of vessel size, vasomotor responses (i.e., vasodilation and vasoconstriction) of retinal arterioles were sensitive to the alpha2-AR antagonist rauwolscine. Consistent with the functional data, alpha2A-AR and eNOS mRNAs were detected in retinal arterioles. Collectively, our data demonstrate that brimonidine at clinical doses evokes a consistent NO-dependent vasodilation in first-order retinal arterioles but a heterogeneous response in second-order arterioles. These vasomotor responses are mediated by the activation of alpha2-AR. It appears that brimonidine, depending on the concentration and vessel size, may alter local retinal blood flow.     

Endogenous and exogenous NO attenuates conduction of vasoconstrictions along arterioles in the microcirculation

Vascular coordination in the microcirculation depends on gap junctional intercellular communication (GJIC), which is reflected by the conduction of locally initiated vasomotor responses. However, little is known about the regulation of GJIC in vivo. We hypothesized that endothelial NO regulates GJIC and therefore studied whether conduction of constrictions and dilations along the vessel wall is modulated by modifying the level of microcirculatory NO. Arterioles were focally stimulated using high K(+) or acetylcholine in the cremaster muscle in situ, and diameter changes were assessed at the local and remote upstream sites by intravital microscopy. Local stimulation with K(+) initiated a constriction that conducted along the arteriole with diminishing amplitude (length constant lambda: 371 +/- 42 mum). After N(omega)-nitro-l-arginine (l-NNA), lambda increased to 507 +/- 30 mum, indicating that GJIC is attenuated by endogenous NO. Exogenous NO, but not adenosine, reduced lambda after l-NNA in a reversible, concentration-dependent, and mainly cGMP-dependent manner as assessed by inhibition of soluble guanylate cyclase. In endothelial NO synthase-deficient mice, lambda was 530 +/- 80 mum and thus similar to that in wild-type mice after l-NNA. Exogenous NO likewise reduced lambda in these mice. The effects of NO were comparable to those of wild-type animals in Cx40-deficient mice, which excludes Cx40 as a specific target of NO. In contrast to constrictions, the amplitude of conducted dilations on acetylcholine did not diminish up to 1,300 mum and were not altered by l-NNA or exogenous NO. We conclude that endogenously released NO attenuates the conduction of vasoconstrictions most likely due to a modulation of gap junctional conductivity. We suggest that this effect is specific for smooth muscle cells, which probably transmit constricting signals, and involves connexins other than Cx40. This mechanism may support the dilatory potency of NO by preventing the conduction of remote vasoconstrictions into areas with basal or activated NO release.     

Are voltage-dependent ion channels involved in the endothelial cell control of vasomotor tone?

In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na(+) (Na(v)) and Ca(2+) channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Na(v) channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Na(v) channel activator veratridine induced an endothelium-dependent dilation. The Na(v) channel isoforms Na(v)1.2, Na(v)1.6, and Na(v)1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Na(v) channels in the conducted response. BAPTA buffering of endothelial cell Ca(2+) delayed and reduced the conducted dilation, which was almost eliminated by Ni(2+), amiloride, or deletion of alpha(1H) T-type Ca(2+) (Ca(v)3.2) channels. Blockade of endothelial nitric oxide synthase or Ca(2+)-activated K(+) channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Na(v) and Ca(v)3.2 channels. The resultant Ca(2+) influx activates endothelial nitric oxide synthase and Ca(2+)-activated K(+) channels, triggering vasodilation.     

Expression of connexin 37, 40 and 43 in rat mesenteric arterioles and resistance arteries

Connexins are the protein constituents of gap junctions which mediate intercellular communication in most tissues. In arterioles gap junctions appear to be important for conduction of vasomotor responses along the vessel. Studies of the expression pattern of connexin isoforms in the microcirculation are sparse. We investigated the expression of the three major vascular connexins in mesenteric arterioles (diameter <50 micro m) from male Sprague-Dawley rats, since conducted vasomotor responses have been described in these vessels. The findings were compared with those obtained from upstream small resistance arteries. Indirect immunofluorescence techniques were used on whole mounts of mesenteric arterioles and on frozen sections of resistance arteries (diameter approximately 300 micro m). Mesenteric arterioles expressed Cx40 and Cx43 in the endothelial layer, and Cx37 was found in most but not all vessels. Connexins were not demonstrated in the media. In resistance arteries endothelial cells expressed Cx37, Cx40 and Cx43. Ultrastructural studies of mesenteric arterioles confirmed that gap junction plaques between endothelial cells are present, whereas myoendothelial, or smooth muscle cell gap junctions could not be demonstrated. The findings suggest that smooth muscle cells in mesenteric arterioles may not be well coupled and favour that conducted vasomotor responses in these vessels are propagated through the endothelial cell layer.     

Participation of intracellular Ca2+ stores in arteriolar conducted responses

We examined the role played by intracellular Ca2+ stores in conducted vasomotor responses induced by phenylephrine (PE) in isolated hamster cremasteric arterioles. When applied briefly ( approximately 1 s) to isolated, cannulated arterioles by using pressure-pulse ejection from a micropipette, PE produced a strong local vasoconstriction and a very small biphasic conducted response (a small constriction followed by a dilation) that propagated several hundred micrometers along the vessel length. The conducted vasomotion was associated with a monophasic elevation of the endothelial cell intracellular Ca2+ concentration ([Ca2+]i) at the site of stimulation, as measured with the Ca2+ indicator fura 2. The Ca2+ pump inhibitor thapsigargin was used to limit filling of Ca2+ stores in smooth muscle and endothelial cells. Thapsigargin reduced baseline diameter and elicited a strong dilator component at the local site while enhancing both the constrictor and dilator components of the PE-induced conducted response. The enhanced conducted constrictor component induced by thapsigargin was mimicked by extraluminal application of tetraethylammonium or charybdotoxin but not by iberiotoxin, apamin, glibenclamide, barium, or 4-aminopirydine. Thapsigargin increased the estimated basal endothelial cell [Ca2+]i by approximately 60 nM and converted the PE-induced change in [Ca2+]i from monotonic to biphasic with a late elevation of [Ca2+]i above baseline that coincided with the increased dilatory component of the conducted response. Luminal application of charybdotoxin plus apamin significantly reduced the dilatory component of the conducted response. These results indicate that intracellular Ca2+ stores play a dynamic role in regulating conducted vasomotor responses apparently through modulation of KCa channels in both cell types.     

Abolition of arteriolar dilation but not constriction to histamine in cremaster muscle of eNOS-/- mice

Histamine increases the permeability of capillaries and venules but little is known of its precapillary actions on the control of tissue perfusion. Using gene ablation and pharmacological interventions, we tested whether histamine could increase muscle blood flow through stimulating nitric oxide (NO) release from microvascular endothelium. Vasomotor responses to topical histamine were investigated in second-order arterioles in the superfused cremaster muscle of anesthetized C57BL6 mice and null platelet endothelial cell adhesion molecule-1 (PECAM-1-/-) and null endothelial NO synthase (eNOS-/-) mice aged 8-12 wk. Neither resting (17 +/- 1 microm) nor maximum diameters (36 +/- 2 microm) were different between groups, nor was the constrictor response (approximately 5 +/- 1 microm) to elevating superfusate oxygen from 0 to 21%. For arterioles of C57BL6 and PECAM-1-/- mice, cumulative addition of histamine to the superfusate produced vasodilation (1 nM-1 microM; peak response, 9 +/- 1 microm) and then vasoconstriction (10-100 microM; peak response, 12 +/- 2 microm). In eNOS-/- mice, histamine produced only vasoconstriction. In C57BL6 and PECAM-1-/- mice, vasodilation was abolished with Nomega-nitro-l-arginine (30 microM); in all mice, vasoconstriction was abolished with nifedipine (1 microM). Vasomotor responses were eliminated with pyrilamine (1 microM; H1 receptor antagonist) yet remained intact with cimetidine (1 microM; H2 receptor antagonist). These findings illustrate that the biphasic vasomotor response of mouse cremaster arterioles to histamine is mediated through H1 receptors on endothelium (NO-dependent vasodilation) as well as smooth muscle (Ca2+ entry and constriction). Thus histamine can increase as well as decrease muscle blood flow, according to local concentration. However, when NO production is compromised, only vasoconstriction and flow reduction occur.     

Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle.

Functional hyperemia requires the coordination of smooth muscle cell relaxation along and between branches of the arteriolar network. Vasodilation is conducted from cell to cell along the arteriolar wall through gap junction channels composed of connexin protein subunits. Within skeletal muscle, it is unclear whether arteriolar endothelium, smooth muscle, or both cell layers provide the cellular pathway for conduction. Furthermore, the constitutive profile of connexin expression within the microcirculation is unknown. We tested the hypothesis that conducted vasodilation and connexin expression are intrinsic to the endothelium of arterioles (17 +/- 1 microm diameter) that supply the skeletal muscle fibers in the cremaster of anesthetized C57BL/6 mice. ACh delivered to an arteriole (500 ms, 1-microA pulse; 1-microm micropipette) produced local dilation of 17 +/- 1 microm; conducted vasodilation observed 1 mm upstream was 9 +/- 1 microm (n = 5). After light-dye treatment to selectively disrupt endothelium (250-microm segment centered 500 microm upstream, confirmed by loss of local response to ACh while constriction to phenylephrine and dilation to sodium nitroprusside remained intact), we found that conducted vasodilation was nearly abolished (2 +/- 1 microm; P < 0.05). Whole-mount immunohistochemistry for connexins revealed punctate labeling at borders of arteriolar endothelial cells, with connexin40 and connexin37 in all branches and connexin43 only in the largest branches. Immunoreactivity for connexins was not apparent in smooth muscle or in capillary or venular endothelium, despite robust immunolabeling for alpha-actin and platelet endothelial cell adhesion molecule-1, respectively. We conclude that vasodilation is conducted along the endothelium of mouse skeletal muscle arterioles and that connexin40 and connexin37 are the primary connexins forming gap junction channels between arteriolar endothelial cells.     

Vasomotor control in arterioles of the mouse cremaster muscle.

Recent advances in transgenic mouse technology provide novel models to study cardiovascular physiology and pathophysiology. In light of these developments, there is an increasing need for understanding cardiovascular function and blood flow control in normal mice. To this end we have used intravital microscopy to investigate vasomotor control in arterioles of the superfused cremaster muscle preparation of anesthetized C57Bl6 mice. Spontaneous resting tone increased with branch order and was enhanced by oxygen. Norepinephrine and acetylcholine (ACh) caused concentration-dependent vasoconstriction and vasodilation, respectively. Microiontophoresis of ACh evoked vasodilation that conducted along arterioles; the local (direct) response was inhibited by N(omega)-nitro-L-arginine (LNA), and both local and conducted responses were inhibited by 17-octadecynoic acid (17-ODYA). Microejection of KCl evoked a biphasic response: a transient conducted vasoconstriction (inhibited by nifedipine), followed by a conducted vasodilation that was insensitive to LNA, indomethacin, and 17-ODYA. Phenylephrine evoked focal vasoconstriction that did not conduct. Perivascular sympathetic nerve stimulation evoked constriction along arterioles that was inhibited by tetrodotoxin. These findings indicate that for arterioles in the mouse cremaster muscle, nitric oxide and endothelial-derived hyperpolarizing factor (as shown by LNA and 17-ODYA interventions, respectively) mediate vasodilatory responses to ACh but not to KCl, and that vasomotor responses spread along arterioles by multiple pathways of cell-to-cell communication.     

Dissection of two Cx37-independent conducted vasodilator mechanisms by deletion of Cx40: electrotonic versus regenerative conduction

Conduction of changes in diameter plays an important role in the coordination of peripheral vascular resistance and, thereby, in the control of arterial blood pressure. It is thought that conduction of vasomotor signals relies on the electrotonic spread of changes in membrane potential from a site of stimulation through gap junctions connecting the cells of the vessel wall. To explore this idea, we stimulated a short segment of mouse cremasteric arterioles with an application, via micropipette, of ACh, an endothelium-dependent vasodilator, or pinacidil, an ATP-sensitive K+ channel opener. Vasodilations were evaluated at the stimulation site (local) and at 500, 1,000, and 2,000 microm upstream. The vasodilator response evoked by direct arteriolar hyperpolarization induced by pinacidil decayed rapidly with distance, as expected for the passive spread of an electrical signal. Deletion of the gap junction proteins connexin37 or connexin40 did not alter the conduction of pinacidil-induced vasodilation. In contrast to pinacidil, the vasodilator response activated by ACh spread along the entire vessel without decrement. Although the ACh-induced conducted vasodilation was similar in wild-type and connexin37 knockout mice, deletion of connexin40 converted the nondecremental conducted response activated by ACh into one similar to that of pinacidil, with a decline in magnitude along the vessel length. These results suggest that ACh activates a mechanism of regenerative conduction of vasodilator responses. Connexin40 is essential for the ACh-activated regenerative vasodilator mechanism. However, neither connexin40 nor connexin37 is indispensable for the electrotonic spread of hyperpolarizing signals.     

Resolution of smooth muscle and endothelial pathways for conduction along hamster cheek pouch arterioles

In the cheek pouch of anesthetized male hamsters, microiontophoresis of Ach (endothelium-dependent vasodilator) or phenylephrine (PE; smooth muscle-specific vasoconstrictor) onto an arteriole (resting diameter, 30-40 microm) evokes vasodilation or vasoconstriction (amplitude, 15-25 microm), respectively, that conducts along the arteriolar wall. In previous studies of conduction, endothelial and smooth muscle layers of the arteriolar wall have remained intact. We tested whether selective damage to endothelium or to smooth muscle would disrupt the initiation and conduction of vasodilation or vasoconstriction. Luminal (endothelial) or abluminal (smooth muscle) light-dye damage was produced within an arteriolar segment centered 500 microm upstream from the distal site of stimulation; conducted responses (amplitude, 10-15 microm) were observed at a proximal site located 1,000 microm upstream. Endothelial damage abolished local responses to ACh in the central segment without affecting those to PE. Nevertheless, ACh delivered at the distal site evoked vasodilation that conducted through the central segment and appeared unhindered at the proximal site. Smooth muscle damage inhibited responses to PE in the central segment and abolished the conduction of vasoconstriction but did not affect conducted vasodilation. We suggest that for cheek pouch arterioles in vivo, vasoconstriction to PE is initiated and conducted within the smooth muscle layer alone. In contrast, once vasodilation to ACh is initiated via intact endothelial cells, the signal is conducted along smooth muscle as well as endothelial cell layers.     

Effects of streptozotocin-induced diabetes on Kv channels in rat small coronary smooth muscle cells

Diabetes impairs endothelium dependent vasodilation, but the mechanism of endothelium independent dilation is not well understood. In the present study, we examined the effect of streptozotocin (STZ)-induced diabetes on the vasomotor of small coronary artery and the activity of voltage-dependent K+ channel of vascular smooth muscle cells in STZ rats [corrected] using the videomicroscopy and patch clamp method. STZ-induced diabetes appeared to [corrected] reduce the vasodilation induced by beta-adrenoceptor agonist, isoproterenol (10(-9)-10(-5) mol/l), and adenylyl cyclase activator forskolin (10(-9)-10(-5) mol/l) respectively (isoproterenol: 44.2 +/- 6.7% vs. 82.5 +/- 4.8%, and forskolin: 54.4 +/- 4.5% vs. 94.3 +/- 2.4%). 4-AP, a Kv channel blocker of VSMC, further decreased dilation to isoproterenol (44.2 +/- 6.7% vs. 10.2 +/- 3.5%) and forskolin (54.4 +/- 4.5% vs. 13.8 +/- 11.0%) significantly. Whole cell K+ current recording demonstrated that STZ-induced diabetes decreased isoproterenol and forskolin-induced K+ current (ISO: 55.6 +/- 7.8 pA/pF vs. 28.4 +/- 3.4 pA/pF, forskolin: 61.3 +/- 9.8 pA/pF vs. 32.4 +/- 3.4 pA/pF). 4-AP further reduced the decreased K+ current (ISO: 28.4 +/- 3.4 pA/pF vs. 14.3 +/- 2.1 pA/pF, forskolin: 32.4 +/- 3.4 pA/pF vs. 14.8 +/- 2.9 pA/pF). These results indicated that STZ-induced diabetes impaired cAMP mediated dilation of small coronary artery and suppressed the Kv channel activity of vascular smooth muscle cells. Kv channel of VSMC was shown to play a determinate role reducing dilation of small coronary artery in STZ rats.     

EDHF,but not NO or prostaglandins,is critical to evoke a conducted dilation upon ACh in hamster arterioles

Vasomotor reactions upon focal stimulation of arterioles have been shown to be conducted along the vascular wall. Such a conduction, which is assumed to reflect the spread of electrical signals, may contribute to coordination of responses within a vascular segment. We aimed to identify which endothelial autacoid(s) act as mediators of the local and conducted dilator responses, respectively. To this end, arterioles in the hamster cremaster microcirculation were locally stimulated with endothelium-dependent [acetylcholine (ACh)] or endothelium-independent dilators [sodium nitroprusside (SNP)], and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.4 mm upstream at distant sites. Experiments were also performed after blockade of nitric oxide (NO) synthase, cyclooxygenase, P-450 monooxygenase, or K(+) channels. Dilations upon ACh (71 +/- 3%) were conducted rapidly (<1 s) to upstream sites (at 1.4 mm: 37 +/- 5%). Although the NO donor SNP induced a similar local dilation (71 +/- 7%), this response was not conducted. Maximal amplitudes of ACh-induced dilations were not attenuated after inhibition of NO synthase and cyclooxygenase at the local and remote sites. However, additional treatment with a P-450 monooxygenase blocker (sulfaphenazole) strongly attenuated the local response (from 62 +/- 9 to 17 +/- 5%) and abrogated dilations at distant sites (at 0.67 mm: from 23 +/- 4% to 4 +/- 3%). Likewise, 17-octadecynoic acid strongly attenuated local and remote responses. Blockers of Ca(2+)-dependent K(+) channels (charybdotoxin or iberiotoxin) attenuated dilations at the local and remote sites after focal application at the ACh stimulation site. In marked contrast, treatment of the upstream site with these blockers was without any effect. We conclude that upon local stimulation with ACh, a cytochrome P-450 monooxygenase product is generated that induces local dilation via the activation of Ca(2+)-dependent K(+) channels and initiates conduction of the dilation. In contrast to the local site, neither activation of these K(+) channels nor the synthesis of NO or prostaglandins is necessary to dilate the arterioles at remote, distant sites. This suggests that endothelium-derived hyperpolarizing factor serves as an important mediator to initiate conducted dilations and, by doing so, may act as a key player in the coordination of arteriolar behavior in the microcirculatory network.     

Impaired nitric oxide synthase-dependent dilatation of cerebral arterioles in type II diabetic rats

The goals of this study were to determine the effects of type II diabetes mellitus on nitric oxide synthase-dependent responses of cerebral arterioles and on endothelial nitric oxide synthase (eNOS) protein in cerebral arterioles. We examined dilatation of cerebral (pial) arterioles in 13-15 week old male lean and diabetic obese Zucker rats in response to nitric oxide synthase-dependent agonists (acetylcholine and adenosine diphosphate (ADP)) and a nitric oxide synthase-independent agonist (nitroglycerin). We found that acetylcholine (10 microM) increased cerebral arteriolar diameter by 10 +/- 3% (mean +/- SE) in lean Zucker rats, but by only 2 +/- 2% in diabetic obese Zucker rats (p<0.05). In addition, ADP (100 microM) increased cerebral arteriolar diameter by 20 +/- 2% in lean Zucker rats, but by only 8 +/- 2% in diabetic obese Zucker rats (p<0.05). In contrast, nitroglycerin produced similar vasodilatation in lean and diabetic obese Zucker rats. Thus, impaired dilatation of cerebral arterioles in diabetic obese Zucker rats is not related to non-specific impairment of vasodilatation. Following these functional studies, we harvested cerebral microvessels for Western blot analysis of eNOS protein. We found that eNOS protein was significantly higher in diabetic obese Zucker rats than in lean Zucker rats (p<0.05). Thus, type II diabetes mellitus impairs nitric oxide synthase-dependent responses of cerebral arterioles. In addition, eNOS protein from cerebral blood vessels is increased in diabetic obese Zucker rats.     

Oxygen distribution in microcirculation after arginine vasopressin-induced arteriolar vasoconstriction

The microvascular distribution of oxygen was studied in the arterioles and venules of the awake hamster window chamber preparation to determine the contribution of vascular smooth muscle contraction to oxygen consumption of the microvascular wall during arginine vasopressin (AVP)-induced vasoconstriction. AVP was infused intravenously at the clinical dosage (0.0001 IU.kg(-1).min(-1)) and caused a significant arteriolar constriction, decreased microvascular flow and functional capillary density, and a substantial rise in arteriolar vessel wall transmural Po(2) difference. AVP caused tissue Po(2) to be significantly lowered from 25.4 +/- 7.4 to 7.2 +/- 5.8 mmHg; however, total oxygen extraction by the microcirculation increased by 25%. The increased extraction, lowered tissue Po(2), and increased wall oxygen concentration gradient are compatible with the hypothesis that vasoconstriction significantly increases vessel wall oxygen consumption, which in this model appears to constitute an important oxygen-consuming compartment. This conclusion was supported by the finding that the small percentage of the vessels that dilated in these experiments had a vessel wall oxygen gradient that was smaller than control and which was not determined by changes in tissue Po(2). These findings show that AVP administration, which reduces oxygen supply by vasoconstriction, may further impair tissue oxygenation by the additional oxygen consumption of the microcirculation.     

Conduction of hyperpolarization along hamster feed arteries: augmentation by acetylcholine

The conduction of vasodilation along resistance vessels has been presumed to reflect the electrotonic spread of hyperpolarization from cell to cell along the vessel wall through gap junction channels. However, the vasomotor response to acetylcholine (ACh) encompasses greater distances than can be explained by passive decay. To investigate the underlying mechanism for this behavior, we tested the hypothesis that ACh augments the conduction of hyperpolarization. Feed arteries (n = 23; diameter, 58 +/- 4 microm; segment length, 2-8 mm) were isolated from the hamster retractor muscle, cannulated at each end, and pressurized to 75 mmHg (at 37 degrees C). Vessels were impaled with one or two dye-containing microelectrodes simultaneously (separation distance, 50 microm to 3.5 mm). Membrane potential (E(m)) (rest, approximately -30 mV) and electrical responses were similar between endothelium and smooth muscle, as predicted for robust myoendothelial coupling. Current injection (-0.8 nA, 1.5 s) evoked hyperpolarization (-10 +/- 1 mV; membrane time constant, 240 ms) that conducted along the vessel with a length constant (lambda) = 1.2 +/- 0.1 mm; spontaneous E(m) oscillations (approximately 1 Hz) decayed with lambda = 1.2 + 0.1 mm. In contrast, ACh microiontophoresis (500 nA, 500 ms, 1 microm tip) evoked hyperpolarization (-14 +/- 2 mV) that conducted with lambda = 1.9 +/- 0.1 mm, 60% further (P < 0.05) than responses evoked by purely electrical stimuli. These findings indicate that ACh augments the conduction of hyperpolarization along the vessel wall.     

TNF-alpha activation of arterioles and venules alters distribution and levels of ICAM-1 and affects leukocyte-endothelial cell interactions

The observation that leukocyte-endothelial cell (EC) interactions are localized to specific regions on the microvessel wall suggests that adhesion molecule distribution is not uniform. We investigated ICAM-1 distribution and leukocyte-EC interactions in blood-perfused microvessels (<80 mum) in cremaster muscle of anesthetized mice, using intravital confocal microscopy and immunofluorescent labeling. Variability of ICAM-1 expression directly determines leukocyte adhesion distribution within the venular microcirculation and contributes to leukocyte rolling in arterioles during inflammation. The number of rolling interactions increased with ICAM-1 intensity (r(2) = 0.69, P < 0.05), and rolling velocity was lower in regions of higher ICAM-1 intensity. In controls, venular ICAM-1 expression was approximately twofold higher than in arterioles. After TNF-alpha treatment, ICAM-1 expression was significantly increased, 2.8 +/- 0.2-fold in arterioles and 1.7 +/- 0.2-fold in venules (P < 0.05). ICAM-1 expression on activated arteriolar ECs only reached the level of control venular ICAM-1. Arteriolar but not venular ECs underwent redistribution of ICAM-1 among cells; some cells increased and some decreased ICAM-1 expression, magnifying the variability of ICAM-1. TNF-alpha treatment increased the length of bright fluorescent regions per unit vessel length (42%, control; 70%, TNF-alpha) along the arteriolar wall, whereas no significant change was observed in venules (60%, control; 63%, TNF-alpha). The spatial distribution and expression levels of adhesion molecules in the microcirculation determine the timing and placement of leukocyte interactions and hence significantly impact the inflammatory response. That arteriolar ECs respond to TNF-alpha by upregulation of ICAM-1, although in a different way compared with venules, suggests an explicit role for arterioles in inflammatory responses.     

Cerebrovascular effects of intravenous dopamine infusions in fetal sheep.

Preterm infants are often treated with intravenous dopamine to increase mean arterial blood pressure (MAP). However, there are few data regarding cerebrovascular responses of developing animals to dopamine infusions. We studied eight near-term and eight preterm chronically catheterized unanesthetized fetal sheep. We measured cerebral blood flow and calculated cerebral vascular resistance (CVR) at baseline and during dopamine infusion at 2.5, 7.5, 25, and 75 microg x kg(-1) x min(-1). In preterm fetuses, MAP increased only at 75 microg x kg(-1) x min(-1) (25 +/- 5%), whereas in near-term fetuses MAP increased at 25 microg x kg(-1) x min(-1) (28 +/- 4%) and further at 75 microg x kg(-1) x min(-1) (51 +/- 3%). Dopamine infusion was associated with cerebral vasoconstriction in both groups. At 25 microg x kg(-1) x min(-1), CVR increased 77 +/- 51% in preterm fetuses and 41 +/- 11% in near-term fetuses, and at 75 microg x kg(-1) x min(-1), CVR increased 80 +/- 33% in preterm fetuses and 83 +/- 21% in near-term fetuses. We tested these responses to dopamine in 11 additional near-term fetuses under alpha-adrenergic blockade (phenoxybenzamine, n = 5) and under dopaminergic D(1)-receptor blockade (SCH-23390, n = 6). Phenoxybenzamine completely blocked dopamine's pressor and cerebral vasoconstrictive effects, while D(1)-receptor blockade had no effect. Therefore, in unanesthetized developing fetuses, dopamine infusion is associated with cerebral vasoconstriction, which is likely an autoregulatory, alpha-adrenergic response to an increase in blood pressure.     

Chronic hyperglycemia impairs functional vasodilation via increasing thromboxane-receptor-mediated vasoconstriction

Individuals with hyperglycemia exhibit impaired exercise performance and functional vasodilatory response. Based on the importance of arachidonic acid (AA) metabolites in functional vasodilation and the increased thromboxane-to-prostacyclin ratio in diabetes, we hypothesized that chronic hyperglycemia in diabetes increases thromboxane-receptor (TP)-mediated vasoconstriction, resulting in an attenuated functional vasodilation. Three groups of lean Zucker rats (8 wk) were used to test the effects of chronic hyperglycemia on endothelial function: normal, streptozotocin (STZ; 70 mg/kg ip), and STZ + insulin (2 U/day). After 4 wk of treatment, spinotrapezius arcade arterioles were chosen for microcirculatory observation. Arteriolar diameter was measured following muscle stimulation and 10 microM AA application in the absence and presence of 1 microM SQ-29548 (TP antagonist). STZ rats exhibited significantly higher fasting glucose levels and attenuated functional and AA-induced dilation compared with normal animals. SQ-29548 improved the vasodilatory responses in STZ rats but had no effect in controls. Insulin treatment normalized both the glucose levels and the vasodilatory responses, and SQ-29548 treatment had no effect on functional or AA-mediated vasodilation in STZ + insulin animals. These results suggest that the impaired functional vasodilation in diabetic rats is due to hyperglycemia-mediated increases in TP-mediated vasoconstriction.