Role of EDHF in conduction of vasodilation along hamster cheek pouch arterioles in vivo

We tested whether local and conducted responses to ACh depend on factors released from endothelial cells (EC) in cheek pouch arterioles of anesthetized hamsters. ACh was delivered from a micropipette (1 s, 500 nA), while arteriolar diameter (rest, approximately 40 microm) was monitored at the site of application (local) and at 520 and 1,040 microm upstream (conducted). Under control conditions, ACh elicited local (22-65 microm) and conducted (14-44 microm) vasodilation. Indomethacin (10 microM) had no effect, whereas N(omega)-nitro-L-arginine (100 microM) reduced local and conducted vasodilation by 5-8% (P < 0.05). Miconazole (10 microM) or 17-octadecynoic acid (17-ODYA; 10 microM) diminished local vasodilation by 15-20% and conducted responses by 50-70% (P < 0.05), suggesting a role for cytochrome P-450 (CYP) metabolites in arteriolar responses to ACh. Membrane potential (E(m)) was recorded in smooth muscle cells (SMC) and in EC identified with dye labeling. At rest (control E(m), typically -30 mV), ACh evoked local (15-32 mV) and conducted (6-31 mV) hyperpolarizations in SMC and EC. Miconazole inhibited SMC and EC hyperpolarization, whereas 17-ODYA inhibited hyperpolarization of SMC but not of EC. Findings indicate that ACh-induced release of CYP metabolites from arteriolar EC evoke SMC hyperpolarization that contributes substantively to conducted vasodilation.     

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.     

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.     

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.     

Contribution of cytochrome P-450 omega-hydroxylase to altered arteriolar reactivity with high-salt diet and hypertension

The present study evaluated the contribution of cytochrome P-450 omega-hydroxylase in modulating the reactivity of cremaster muscle arterioles in normotensive rats on high-salt (HS) and low-salt (LS) diet and in rats with reduced renal mass hypertension (RRM-HT). Changes in arteriolar diameter in response to ACh, sodium nitroprusside (SNP), ANG II, and elevated O(2) were measured via television microscopy under control conditions and following cytochrome P-450 omega-hydroxylase inhibition with 17-octadecynoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In normotensive rats on either LS or HS diet, resting tone was unaffected and arteriolar reactivity to ACh or SNP was minimally affected by cytochrome P-450 omega-hydroxylase inhibition. In RRM-HT rats, cytochrome P-450 omega-hydroxylase inhibition reduced resting tone and significantly enhanced arteriolar dilation to ACh and SNP. Treatment with 17-ODYA or DDMS inhibited arteriolar constriction to ANG II and O(2) in all the groups, although the degree of inhibition was greater in RRM-HT than in normotensive animals. These results suggest that metabolites of cytochrome P-450 omega-hydroxylase contribute to the altered reactivity of skeletal muscle arterioles to vasoconstrictor and vasodilator stimuli in RRM-HT.     

Endothelial cell signaling during conducted vasomotor responses

ACh and KCl stimulate vasomotor responses that spread rapidly and bidirectionally along arteriole walls, most likely via spread of electric current or Ca2+ through gap junctions. We examined these possibilities with isolated, cannulated, and perfused hamster cheek pouch arterioles (50- to 80-microm resting diameter). After intraluminal loading of 2 microM fluo 3 to measure Ca2+ or 1 microM di-8-ANEPPS to measure membrane potential, photometric techniques were used to selectively measure changes in intracellular Ca2+ concentration ([Ca2+]i) or membrane potential in endothelial cells. Activation of the endothelium by micropipette application of ACh (10-4 M, 1.0-s pulse) to a short segment of arteriole (100-200 microm) increased endothelial cell [Ca2+]i and caused hyperpolarization at the site of stimulation. This response was followed rapidly by vasodilation of the entire arteriole ( approximately 2-mm length). Change in membrane potential always preceded dilation, both at the site of stimulation and at distant sites along the arteriole. In contrast, an increase in endothelial cell [Ca2+]i was observed only at the application site. Micropipette application of KCl, which can depolarize both smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a rapid, spreading response consisting of depolarization followed by vasoconstriction. With KCl stimulation, in addition to changes in membrane potential, increases in endothelial cell [Ca2+]i were observed at distant sites not directly exposed to KCl. The rapid longitudinal spread of both hyperpolarizing and depolarizing responses support electrical coupling as the mode of signal transmission along the arteriolar length. In addition, the relatively short distance between heterologous cell types enables the superimposed radial Ca2+ signaling between smooth muscle and endothelial cells to modulate vasomotor responses.     

Evidence for impaired neurovascular transmission in a murine model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by mutations in the dystrophin gene. Little is known about how blood flow control is affected in arteriolar networks supplying dystrophic muscle. We tested the hypothesis that mdx mice, a murine model for DMD, exhibit defects in arteriolar vasomotor control. The cremaster muscle was prepared for intravital microscopy in pentobarbital sodium-anesthetized mdx and C57BL/10 control mice (n ≥ 5 per group). Spontaneous vasomotor tone increased similarly with arteriolar branch order in both mdx and C57BL/10 mice [pooled values: first order (1A), 6%; second order (2A), 56%; and third order (3A), 61%] with no difference in maximal diameters between groups measured during equilibration with topical 10 μM sodium nitroprusside (pooled values: 1A, 70 ± 3 μm; 2A, 31 ± 3 μm; and 3A, 19 ± 3 μm). Concentration-response curves to acetylcholine (ACh) and norepinephrine added to the superfusion solution did not differ between mdx and C57BL/10 mice, nor did constriction to elevated (21%) oxygen. In response to local stimulation from a micropipette, conducted vasodilation to ACh and conducted vasoconstriction to KCl were also not different between groups; however, constriction decayed with distance (P < 0.05) whereas dilation did not. Remarkably, arteriolar constriction to perivascular nerve stimulation (PNS) at 2, 4, and 8 Hz was reduced by ～25-30% in mdx mice compared with C57BL/10 mice (P < 0.05). With intact arteriolar reactivity to agonists, attenuated constriction to perivascular nerve stimulation indicates impaired neurovascular transmission in arterioles controlling blood flow in mdx mice.     

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.     

Streptozotocin-induced Diabetes Decreases Conducted Vasoconstrictor Response in Mouse Cremaster Arterioles

A conducted vasomotor response (CVR) is characterized by the spread of vasoconstriction or vasodilatation both up- and downstream from a local stimulation site in the microcirculation. It is believed to coordinate vasomotor responses within the microcirculation, and to contribute to the control of the major feed arteries to a given organ or tissue. Microvascular disease is a common and severe complication in diabetes, and we therefore studied CVR in streptozotocin (STZ) diabetic mice to examine whether changes in CVR might have a role in the pathophysiology of microvascular dysfunction in diabetes. The mouse cremasteric arterioles were stimulated locally with KCl and the resulting local response as well as conducted responses at 500 mum and 1000 mum were measured in control and STZ treated mice. Diabetes (n=8) induced by intraperitoneal injection of STZ in a dose of 100 mg/kg (mean blood glucose 16.8+/-2.1 mmol/l) decreased the conduction of vasoconstriction from 27.3+/-1.1% to 21.4+/-1.6% at 500 mum (p<0.01) and from 17.4+/-1.0% to 9.8+/-1.1% at 1000 mum (p<0.01) as compared with control (n=9). Treatment with either the protein kinase C beta II inhibitor (LY341684) or the oxygen radical scavenger tempol, did not improve the decreased conduction of vasoconstriction, but when administered together, the conduction of vasoconstriction was improved from 21.4+/-1.6% to 26.5+/-0.8% at 500 mum and 9.8+/-1.1% to 16.5+/-0.7% at 1000 mum (p<0.01). We conclude that STZ induced diabetes reduces conducted vasoconstriction to KCl in mouse cremasteric arterioles, and combined treatment with both an oxygen radical scavenger and a protein kinase C beta II inhibitor improves the reduced conducted vasoconstriction.     

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.     

Improved functional vasodilation in obese Zucker rats following exercise training

Obese individuals exhibit impaired functional vasodilation and exercise performance. We have demonstrated in obese Zucker rats (OZ), a model of morbid obesity, that insulin resistance impairs functional vasodilation via an increased thromboxane receptor (TP)-mediated vasoconstriction. Chronic treadmill exercise training improves functional vasodilation in the spinotrapezius muscle of the OZ, but the mechanisms responsible for the improvement in functional vasodilation are not clear. Based on evidence that exercise training improves insulin resistance, we hypothesized that, in the OZ, exercise training increases functional vasodilation and exercise capability due to decreases TP-mediated vasoconstriction associated with improved insulin sensitivity. Six-week-old lean Zucker rats (LZ) and OZ were exercised on a treadmill (24 m/min, 30 min/day, 5 days/wk) for 6 wk. An oral glucose tolerance test was performed at the end of the training period. We measured functional vasodilation in both exercise trained (spinotrapezius) and nonexercise trained (cremaster) muscles to determine whether the improved functional vasodilation following exercise training in OZ is due to a systemic improved insulin resistance. Compared with LZ, the sedentary OZ exhibited impairments in glucose tolerance and functional vasodilation in both muscles. The TP antagonist SQ-29548 improved the vasodilator responses in the sedentary OZ with no effect in the LZ. Exercising training of the LZ increased the functional vasodilation in spinotrapezius muscle, with no effect in the cremaster muscle. Exercising training of the OZ improved glucose tolerance, along with increased functional vasodilation, in both the spinotrapezius and cremaster muscles. SQ-29548 treatment had no effect on the vasodilator responses in either cremaster or spinotrapezius muscles of the exercise-trained OZ. These results suggest that, in the OZ, there is a global effect of exercising training to improve insulin resistance and increase functional vasodilation via a decreased TP-mediated vasoconstriction.     

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.     

Time course of flow-induced vasodilation in skeletal muscle: contributions of dilator and constrictor mechanisms

The purpose of this study was to determine the time course of flow-induced vasodilation in soleus and gastrocnemius muscle arterioles and the mechanisms that underlie vasodilatory responses to an increase in intraluminal flow. Vasodilation was assessed during 20 min of continuous exposure to intraluminal flow. Both soleus and gastrocnemius muscle arterioles dilated in response to flow, although the magnitude of vasodilation was greater in arterioles from the gastrocnemius muscle. Neither blockade of nitric oxide synthase with N(G)-nitro-L-arginine methyl ester (L-NAME) nor blockade of cyclooxygenase with indomethacin inhibited the initial vasodilation (0-2 min) in arterioles from either muscle. In contrast, vasodilation to sustained exposure to flow (2-20 min) was eliminated by treatment with L-NAME in arterioles from both muscles. Both depolarization with 40 mM KCl and blockade of Ca(2+)-activated K(+) channels inhibited the initial flow-induced dilation, and the inhibition was greater in gastrocnemius muscle arterioles than soleus muscle arterioles. In the presence of L-NAME, prolonged exposure to flow resulted in constriction in soleus and gastrocnemius muscle arterioles. This constriction was abolished by endothelin receptor blockade. These results indicate that the time course and magnitude of flow-induced vasodilation differs between arterioles from soleus and gastrocnemius muscles. The immediate response to increased flow is greater in gastrocnemius muscle arterioles and involves activation of K(+) channels. In arterioles from both soleus and gastrocnemius muscles, vasodilation to sustained flow exposure occurs primarily through production of nitric oxide. In the absence of nitric oxide, sustained exposure to flow results in pronounced constriction that is mediated by endothelin.     

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.     

Dietary obesity increases NO and inhibits BKCa-mediated, endothelium-dependent dilation in rat cremaster muscle artery: association with caveolins and caveolae

Obesity is a risk factor for hypertension and other vascular disease. The aim of this study was to examine the effect of diet-induced obesity on endothelium-dependent dilation of rat cremaster muscle arterioles. Male Sprague-Dawley rats (213 ± 1 g) were fed a cafeteria-style high-fat or control diet for 16-20 wk. Control rats weighed 558 ± 7 g compared with obese rats 762 ± 12 g (n = 52-56; P < 0.05). Diet-induced obesity had no effect on acetylcholine (ACh)-induced dilation of isolated, pressurized (70 mmHg) arterioles, but sodium nitroprusside (SNP)-induced vasodilation was enhanced. ACh-induced dilation of arterioles from control rats was abolished by a combination of the K(Ca) blockers apamin, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), and iberiotoxin (IBTX; all 0.1 μmol/l), with no apparent role for nitric oxide (NO). In arterioles from obese rats, however, IBTX had no effect on responses to ACh while the NO synthase (NOS)/guanylate cyclase inhibitors N(ω)-nitro-L-arginine methyl ester (L-NAME; 100 μmol/l)/1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μmol/l) partially inhibited ACh-induced dilation. Furthermore, NOS activity (but not endothelial NOS expression) was increased in arteries from obese rats. L-NAME/ODQ alone or removal of the endothelium constricted arterioles from obese but not control rats. Expression of caveolin-1 and -2 oligomers (but not monomers or caveolin-3) was increased in arterioles from obese rats. The number of caveolae was reduced in the endothelium of arteries, and caveolae density was increased at the ends of smooth muscle cells from obese rats. Diet-induced obesity abolished the contribution of large-conductance Ca(2+)-activated K(+) channel to ACh-mediated endothelium-dependent dilation of rat cremaster muscle arterioles, while increasing NOS activity and inducing an NO-dependent component.     

Rostral Ventrolateral Medulla: An Integrative Site for Muscle Vasodilation During Defense-Alerting Reactions

1. Evidence gathered over the last 30 years has firmly established that the rostral ventrolateral medulla (RVLM) is a major vasomotor center in the brainstem, harboring sympathetic premotor neurons responsible for generating and maintaining basal vasomotor tone and resting levels of arterial blood pressure. Although the RVLM has been almost exclusively classified as a vasopressor area, in this report we review some evidence suggesting a prominent role of the RVLM in muscle vasodilation during defense-alerting responses.2. Defense-alerting reactions are a broad class of behavior including flexion of a limb, fight/flight responses, apologies, etc. They comprise species-distinctive motor and neurovegetative adjustments. Cardiovascular responses include hypertension, tachycardia, visceral vasoconstriction, and muscle vasodilation. Since defense-alerting reactions generally involve intense motor activation, muscle vasodilation is regarded as a key feature of these responses.3. In anesthetized or unanesthetized-decerebrate animals, natural or electrical stimulation of cutaneous and muscle afferents produced hypertension, tachycardia, and vasodilation restricted to the stimulated limb.4. Unilateral inactivation of the RVLM contralateral to the stimulated limb abolished cardiovascular adjustments to stimulation of cutaneous and muscle afferents. Within the RVLM glutamatergic synapses mediate pressor responses, whereas GABAergic synapses mediates muscle vasodilation.5. In urethane-anesthetized rats, electrical stimulation of the hypothalamus elicited hypertension, tachycardia, visceral vasoconstriction, and hindlimb vasodilation. The hindlimb vasodilation induced by hypothalamic stimulation is a complex response, involving reduction of sympathetic vasoconstrictor tone, release of catecholamines by the adrenal medulla, and a still unknown system that may use nitric oxide as a mediator.6. Blockade of glutamatergic transmission within the RVLM selectively blocks muscle vasodilation induced by hypothalamic stimulation.7. The results obtained suggest that, besides its role in the generation and maintenance of the sympathetic vasoconstrictor drive, the RVLM is also critical for vasodilatory responses during defense reactions. The RVLM may contain several, distinctive mechanisms for muscle vasodilation. Anatomical and functional characterization of these pathways may represent a breakthrough in our understanding of cardiovascular control in normal and/or pathological conditions.     

Remote arteriolar dilations in response to muscle contraction under capillaries

In hamster cremaster muscle, it has been shown previously that contraction of skeletal muscle fibers underlying small groups of capillaries (modules) induces dilations that are proportional to metabolic rate in the two arteriolar generations upstream of the stimulated capillaries (Berg BR, Cohen KD, and Sarelius IH. Am J Physiol Heart Circ Physiol 272: H2693-H2700, 1997). These remote dilations were hypothesized to be transmitted via gap junctions and not perivascular nerves. In the present study, halothane (0.07%) blocked dilation in the module inflow arteriole, and dilation in the second arteriolar generation upstream, the branch arteriole, was blocked by both 600 mosM sucrose and halothane but not tetrodotoxin (2 microM). Dilations in both arterioles were not blocked by the gap junction uncoupler 18-beta-glycyrrhetinic acid (40 microM), and 80 mM KCl did not block dilation of the module inflow arteriole. These data implicate a gap junctional-mediated pathway insensitive to 18-beta-glycyrrhetinic acid in dilating the two arterioles upstream of the capillary module during "remote" muscle contraction. Dilation in the branch arteriole, but not the module inflow arteriole, was attenuated by 100 microM N(omega)-nitro-L-arginine. Thus selective contraction of muscle fibers underneath capillaries results in dilations in the upstream arterioles that have characteristics consistent with a signal that is transmitted along the vessel wall through gap junctions, i.e., a conducted vasodilation. The observed insensitivities to 18-beta-glycyrrhetinic acid, to KCl, and to N(omega)-nitro-L-arginine suggest, however, that there are multiple signaling pathways by which remote dilations can be initiated in these microvessels.     

Aging impairs endothelium-dependent vasodilation in rat skeletal muscle arterioles

Blood flow capacity in skeletal muscle declines with age. Reduced blood flow capacity may be related to decline in the maximal vasodilatory capacity of the resistance vasculature. This study tested the hypothesis that aging results in impaired vasodilatory capacity of first-order (1A) arterioles isolated from rat-hindlimb locomotory muscle: 1A arterioles (90-220 microm) from gastrocnemius and soleus muscles of young (4 mo) and aged (24 mo) Fischer-144 rats were isolated, cannulated, and pressurized via hydrostatic reservoirs. Vasodilatory responses to increasing concentrations of ACh (10(-9) to 10(-4) M), adenosine (ADO, 10(-10) to 10(-4) M), and sodium nitroprusside (SNP, 10(-10) to 10(-4) M) were evaluated at a constant intraluminal pressure of 60 cmH(2)O in the absence of flow. Flow-induced vasodilation was also evaluated in the absence of pressure changes. Responses to ADO and SNP were not altered by age. Endothelium-dependent vasodilation induced by flow was significantly reduced in arterioles from both gastrocnemius and soleus muscles. In contrast, endothelium-dependent vasodilation to ACh was reduced only in soleus muscle arterioles. These results indicate that aging impairs vasodilatory responses mediated through the endothelium of resistance arterioles from locomotory muscle, whereas smooth muscle vasodilatory responses remain intact with aging. Additionally, ACh-induced vasodilation was altered by age only in soleus muscle arterioles, suggesting that the mechanism of age-related endothelial impairment differs in arterioles from soleus and gastrocnemius muscles.     

Arteriolar reactivity in lymphocyte-deficient mice

Mounting evidence suggests that lymphocytes have the capacity to contribute to the regulation of systemic circulatory control. We postulated that T and natural killer (NK) cells could modify basal microvascular activity under physiologically normal conditions. In situ intravital microscopy of mouse cremaster vasculature was used to evaluate arteriolar reactivities to the vasoconstrictors angiotensin II (ANG II) and phenylephrine (Phe) and the vasodilators acetylcholine (ACh) and adenosine (Ado) in normal [+/+; wild type (WT)] and genetically immunodeficient (T(-)B(-)NK(+) or T(-)B(-)\NK(-)) C57BL/6 and BALB/c mice, strain backgrounds with differentially polarized T cell cytokine production. Immunodeficient mice tended to have smaller baseline and maximal diameters of third-order cremaster arterioles than their congenic WT partners. In C57BL/6, baseline diameters were similar in T-B(-) mice without or with NK cells; in BALB/c, baseline diameters were larger in T-B-NK(-) mice than in T(-)B(-)NK(+) mice. Thus, at baseline, lymphocytes tended to promote vasodilation, except BALB/c NK cells, which mediated mild vasoconstriction. The presence of NK cells suppressed dilations to Ado in both strains, to ACh in the C57BL/6 strain, and dilatory responses to ANG II in C57BL/6 and to Phe in BALB/c. In the BALB/c strain, the presence of T and B cells promoted vasodilatory responses to Ado, attenuated dilations to low ACh concentrations, and exaggerated dilation and constriction responses to ANG II. Thus, under agonist challenge, NK cells generally promote constriction, whereas influences of T and B cells depend upon the stimulus. Therefore, lymphocytes or their products have physiological influences on microvascular arteriolar reactivity.