Cheek Pouch Arterioles Research Articles

NRF2 activation with Protandim attenuates salt-induced vascular dysfunction and microvascular rarefaction.

This study tested the hypothesis that dietary activation of the master antioxidant and cell protective transcription factor nuclear factor, erythroid -2-like 2 (NRF2), protects against salt-induced vascular dysfunction by restoring redox homeostasis in the vasculature. Male Sprague-Dawley rats and Syrian hamsters were fed a HS (4.0% NaCl) diet containing ~60 mg/kg/day Protandim supplement for 2 weeks and compared to controls fed HS diet alone. Protandim supplementation restoredendothelium-dependent vasodilation in response to acetylcholine (ACh) in middle cerebral arteries (MCA)of HS-fed rats and hamster cheek pouch arterioles, and increased microvessel density in the cremastermuscle of HS-fed rats. The restored dilation to ACh in MCA of Protandim-treated rats was prevented by inhibiting nitric oxide synthase (NOS) with L-NAME [100 μM] and was absent in MCA from Nrf2(-/-) knockout rats fed HS diet. Basilar arteries from HS-fed rats treated with Protandim exhibited significantly lower staining for mitochondrial oxidizing species than untreated animals fed HS diet alone; and Protandim treatment increased MnSOD (SOD2) protein expression in mesenteric arteries of HS-fed rats. These results suggest that dietary activation of NRF2 protects against salt-induced vascular dysfunction, vascular oxidative stress, and microvascular rarefaction by upregulating antioxidant defenses and reducing mitochondrial ROS levels.

Length adaptation of smooth muscle contractile filaments in response to sustained activation

Airway and bladder smooth muscles are known to undergo length adaptation under sustained contraction. This adaptation process entails a remodelling of the intracellular actin and myosin filaments which shifts the peak of the active force–length curve towards the current length. Smooth muscles are therefore able to generate the maximum force over a wide range of lengths. In contrast, length adaptation of vascular smooth muscle has attracted very little attention and only a handful of studies have been reported. Although their results are conflicting on the existence of a length adaptation process in vascular smooth muscle, it seems that, at least, peripheral arteries and arterioles undergo such adaptation. This is of interest since peripheral vessels are responsible for pressure regulation, and a length adaptation will affect the function of the cardiovascular system. It has, e.g., been suggested that the inward remodelling of resistance vessels associated with hypertension disorders may be related to smooth muscle adaptation. In this study we develop a continuum mechanical model for vascular smooth muscle length adaptation by assuming that the muscle cells remodel the actomyosin network such that the peak of the active stress–stretch curve is shifted towards the operating point. The model is specialised to hamster cheek pouch arterioles and the simulated response to stepwise length changes under contraction. The results show that the model is able to recover the salient features of length adaptation reported in the literature.

Reduced angiotensin II levels cause generalized vascular dysfunction via oxidant stress in hamster cheek pouch arterioles

ObjectivesWe investigated the effect of suppressing plasma angiotensin II (ANG II) levels on arteriolar relaxation in the hamster cheek pouch. MethodsArteriolar diameters were measured via television microscopy during short-term (3–6days) high salt (HS; 4% NaCl) diet and angiotensin converting enzyme (ACE) inhibition with captopril (100mg/kg/day). ResultsACE inhibition and/or HS diet eliminated endothelium-dependent arteriolar dilation to acetylcholine, endothelium-independent dilation to the NO donor sodium nitroprusside, the prostacyclin analogs carbacyclin and iloprost, and the KATP channel opener cromakalim; and eliminated arteriolar constriction during KATP channel blockade with glibenclamide. Scavenging of superoxide radicals and low dose ANG II infusion (25ng/kg/min, subcutaneous) reduced oxidant stress and restored arteriolar dilation in arterioles of HS-fed hamsters. Vasoconstriction to topically-applied ANG II was unaffected by HS diet while arteriolar responses to elevation of superfusion solution PO2 were unaffected (5% O2, 10% O2) or reduced (21% O2) by HS diet. ConclusionsThese findings indicate that sustained exposure to low levels of circulating ANG II leads to widespread dysfunction in endothelium-dependent and independent vascular relaxation mechanisms in cheek pouch arterioles by increasing vascular oxidant stress, but does not potentiate O2- or ANG II-induced constriction of arterioles in the distal microcirculation of normotensive hamsters.

Connexin Isoform Expression in Smooth Muscle Cells and Endothelial Cells of Hamster Cheek Pouch Arterioles and Retractor Feed Arteries

Gap junction channels formed by connexin (Cx) protein subunits enable cell-to-cell conduction of vasoactive signals. Given the lack of quantitative measurements of Cx expression in microvascular endothelial cells (EC) and smooth muscle cells (SMC), the objective was to determine whether Cx expression differed between EC and SMC of resistance microvessels for which conduction is well-characterized. Cheek pouch arterioles (CPA) and retractor feed arteries (RFA) were hand-dissected and dissociated to obtain SMC or endothelial tubes. In complementary experiments, small intestine was dissociated to obtain SMC. Following reverse transcription, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed by using specific primers and fluorescent probes for Cx37, Cx40, and Cx43. Smooth muscle alpha-actin (SMAA) and platelet endothelial cell adhesion molecule-1 (PECAM-1) served as respective reference genes. Transcript copy numbers were similar for each Cx isoform in EC from CPA and RFA (approximately 0.5 Cx/PECAM-1). For SMC, Cx43 transcript in CPA and RFA (< 0.1 Cx/SMAA) was less (p < 0.05) than that in small intestine (approximately 0.4 Cx/SMAA). Transcripts for Cx37 and Cx40 were also detected in SMC. Punctate immunolabeling for each Cx isoform was pronounced at EC borders and that for Cx43 was pronounced in SMC of small intestine. In contrast, Cx immunolabeling was not detected in SMC of CPA or RFA. Connexin expression occurs primarily within the endothelium of arterioles and feed arteries, supporting a highly effective pathway for conducting vasoactive signals along resistance networks. The apparent paucity of Cx expression within SMC underscores discrete homocellular coupling and focal localization of myoendothelial gap junctions.

Connexin isoform expression in microvascular smooth muscle and endothelium

Endothelial cells (EC) have been implicated in providing the pathway for conducted vasodilation. A complementary role for smooth muscle cells (SMC) remains controversial, in part due to lack of quantitative measurements of Cx expression in respective cell layers. Our goal here was to quantify the expression of Cx isoforms in EC and SMC from hamster cheek pouch arterioles (CPA) and retractor feed arteries (RFA). Microvessels were dissected, dissociated, and SMC (300–400 cells) or EC (6–10 tubes) were collected to generate cDNA. Following reverse transcription, qRT-PCR was performed for Cx37, Cx40, Cx43, PECAM-1 and smooth muscle α-actin (SMAA). Transcript copy numbers were similar for each Cx isoform in EC from CPA and RFA (~0.5 Cx/PECAM-1) and relatively higher (P<0.05) than those in SMC (<0.1 Cx/SMAA). In contrast, Cx43 transcript in SMC from small intestine approached levels in EC (~0.4 Cx43/SMAA). Punctate immunolabeling for Cx was robust at cell borders of EC tubes and intestinal SMC but undetectable in SMC of CPA or RFA. We conclude that EC are the principal site of Cx expression in CPA and RFA. Low Cx expression in SMC may reflect discrete homocellular and myoendothelial gap junctions. (Support: HL41026, HL56786, HL32469).

Hypoxia-induced vascular smooth muscle relaxation: increased ATP-sensitive K+ efflux or decreased voltage-sensitive Ca2+ influx?

controversy continues regarding the cellular mechanisms of hypoxic vasodilation. The vascular endothelium may contribute to hypoxic dilations by releasing relaxing factors that act on the smooth muscle ([4][1], [6][2]–[8][3]). In contrast, numerous studies have suggested a role of the vascular

Functional activity of BKCa channels is not coupled to the activity of ryanodine receptors in hamster cheek pouch arterioles

The activity of large conductance, calcium-activated potassium (BKCa) channels appears to be tightly coupled to the activity of ryanodine receptors (RYR) in vascular smooth muscle (VSM) cells in myogenically active arteries. In these vessels, blockade of BKCa channels or RYR causes equivalent membrane depolarization and constriction supporting this hypothesis. However, the linkage between BKCa channels and RYR in arterioles has not been established. Hamster cheek pouch arterioles (maximum diameter = 87 μm) were isolated, cannulated with glass micropipettes, pressurized to 70 cm H2O, and internal diameter measured as an index of VSM cell contractile activity. At rest, these arterioles developed substantial myogenic tone: resting diameters at 70 cm H2O were 53 ± 4 μm largely due to Ca2+ influx through L-type calcium channels. Superfusion with 1 mM tetraethyl ammonium (TEA) to block BKCa channels, constricted the arterioles 14 ± 3 μm (n = 15, P < 0.05). In contrast, superfusion of arterioles (resting diameter = 44 ± 3 μm) with 10 μM ryanodine had no significant effect: the vessels constricted 1 ± 1 micrometer (n = 7, P > 0.05). In these same vessels, in the presence of ryanodine, 1 mM TEA constricted the arterioles 7 ± 2 μm (n = 7, P < 0.05). These data indicate that the activity of BKCa channels is not coupled to the activity of RYR receptors in hamster cheek pouch arteriolar smooth muscle cells. Based on our previous observations, we speculate that it is Ca2+ influx through L-type voltage-gated Ca2+ channels that is responsible for active BKCa channels in microvascular smooth muscle cells. Supported by PHS grant HL32469.

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.

Homocellular conduction along endothelium and smooth muscle of arterioles in hamster cheek pouch: unmasking an NO wave.

We investigated roles for homocellular (endothelium or smooth muscle) and heterocellular (myoendothelial) conduction pathways along hamster cheek pouch arterioles in vivo (n=64; diameter, 33+/-1 microm). Endothelium-dependent and -independent vasoactive agents were delivered from micropipettes (0.5 or 1 second pulse) onto an arteriole while observing diameter changes at defined distances along the vessel. Acetylcholine (ACh) produced maximal diameter (63+/-1 microm) locally and vasodilation conducted rapidly ( approximately 10 microm response at 2 mm, <1 second). Responses to bradykinin (BK) were similar, whereas sodium nitroprusside produced maximal dilation locally without conduction. KCl evoked biphasic conduction of vasoconstriction and vasodilation, whereas phenylephrine (PE) produced conducted vasoconstriction. Disrupting the integrity of endothelium as a conduction pathway using focal light-dye treatment (LDT) abolished conducted vasodilation to BK and to KCl but not to ACh. Disruption of smooth muscle integrity with LDT abolished conducted vasoconstriction with no effect on conducted vasodilation. After LDT of respective cell layers at sites 1 mm apart, vasodilation to ACh conducted past disrupted smooth muscle or disrupted endothelium, but not beyond both sites in series. The loss of conduction after selective LDT indicates a lack of effective myoendothelial coupling along the arteriolar wall. During NO synthase inhibition (L-NA, 100 micromol/L), conducted vasodilation was abolished to BK and to KCl yet remained intact to ACh. However, after LDT of smooth muscle, L-NA inhibited conduction to ACh by 60%. Thus, conduction of vasodilation entails a wave of NO release along arteriolar endothelium that is masked when smooth muscle provides a parallel conduction pathway.

Arteriolar occlusion causes independent cellular responses in endothelium and smooth muscle.

To test the hypothesis that arteriolar occlusion causes different cellular changes in endothelial and smooth muscle cells. Cheek pouch arterioles (resting diameter 41 +/- 2 microm) of anesthetized hamsters were occluded briefly (<60 seconds) either upstream or downstream from an observation site. Changes in membrane potential and intracellular calcium concentration ([Ca(2+)](i)) of the endothelial or smooth muscle cells were determined by using fluorescence microscopy (ratiometric analysis). The pressure in the occluded segment decreased by 17.4 +/- 2.6 cm H(2)O during upstream occlusion and increased by 16.8 +/- 6 cm H(2)O during downstream occlusion (n = 5). Upstream occlusion caused vasoconstriction of the occluded segment by 2.4 +/- 0.4 microm, whereas downstream occlusion produced brief vasodilatation by 1.1 +/- 0.2 microm. The endothelial cells hyperpolarized during upstream or downstream occlusion (ratio change: 2.26 +/- 0.24% and 2.39 +/- 0.42%, respectively; p < 0.01, n = 5). There were no changes in endothelial [Ca(2+)](i). The smooth muscle cells depolarized (ratio change: -2.08 +/- 0.14%, n = 5) with an increase in [Ca(2+)](i) (ratio change: 2.92 +/- 0.16%, n = 6) during downstream occlusion. However, there was no detectable change in membrane potential or [Ca(2+)](i) of smooth muscle cells during upstream occlusion. All the changes rapidly recovered when occlusion was released. Responses of an in-situ isolated segment on a side branch revealed conducted dilatory signals caused by the occlusions. Our results show that the endothelial and smooth muscle cells respond independently to arteriolar occlusion. The endothelial and smooth muscle cells do not effectively communicate in [Ca(2+)](i) or membrane potential during acute arteriolar occlusion. Hyperpolarizing signals in endothelium cause conducted dilation.

Acute and chronic treatment with nicotine impairs reactivity of arterioles in response to activation of potassium channels.

Although acute and chronic treatment with nicotine impairs nitric oxide synthase-dependent responses of large and small blood vessels, the effect of nicotine on other vasodilator pathways remains uncertain. The goal of the current study was to determine effects of nicotine on dilatation of arterioles to activation of ATP-sensitive potassium channels. Reactivity of cheek pouch arterioles ( approximately 50 microm) was measured during acute (1-2 h) and chronic (2-to 3-week) exposure to nicotine in response to aprikalim, cromakalim, and nitroglycerin. Acute treatment with nicotine impaired dilatation of arterioles in response to aprikalim and cromakalim but not nitroglycerin. Aprikalim and cromakalim (1.0 microM) dilated arterioles by 37 +/- 5% and 30 +/- 3%, respectively, before, but by only 21 +/- 4% and 16 +/- 3%, respectively, after infusion of nicotine (p < 0.05). Chronic exposure to nicotine did not alter vasodilatation to nitroglycerin but impaired vasodilatation to aprikalim and cromakalim. In vehicle-treated hamsters, aprikalim and cromakalim (1.0 microM) dilated arterioles by 28 +/- 1% and 32 +/- 3%, respectively. However, in nicotine-treated hamsters aprikalim and cromakalim (1.0 microM) dilated arterioles by only 3 +/- 1% and 13 +/- 1%, respectively. Next, the role of superoxide anion in impaired responses of arterioles to aprikalim and cromakalim during acute infusion of nicotine was examined. Treatment with superoxide dismutase attenuated the effects of nicotine on aprikalim and cromakalim. Thus, acute and chronic exposure to nicotine has profound affects on vasodilatation to activation of ATP-sensitive potassium channels, which may be mediated by superoxide anion.

Arteriolar Occlusion Causes Independent Cellular Responses in Endothelium and Smooth Muscle

Objectives: To test the hypothesis that arteriolar occlusion causes different cellular changes in endothelial and smooth muscle cells. Methods: Cheek pouch arterioles (resting diameter 41 ± 2 µm) of anesthetized hamsters were occluded briefly (<60 seconds) either upstream or downstream from an observation site. Changes in membrane potential and intracellular calcium concentration ([Ca2+]i) of the endothelial or smooth muscle cells were determined by using fluorescence microscopy (ratiometric analysis). Results: The pressure in the occluded segment decreased by 17.4 ± 2.6 cm H2O during upstream occlusion and increased by 16.8 ± 6 cm H2O during downstream occlusion (n= 5). Upstream occlusion caused vasoconstriction of the occluded segment by 2.4 ± 0.4 µm, whereas downstream occlusion produced brief vasodilatation by 1.1 ± 0.2 µm. The endothelial cells hyperpolarized during upstream or downstream occlusion (ratio change: 2.26 ± 0.24% and 2.39 ± 0.42%, respectively; p < 0.01, n= 5). There were no changes in endothelial [Ca2+]i. The smooth muscle cells depolarized (ratio change: −2.08 ± 0.14%, n= 5) with an increase in [Ca2+]i (ratio change: 2.92 ± 0.16%, n= 6) during downstream occlusion. However, there was no detectable change in membrane potential or [Ca2+]i of smooth muscle cells during upstream occlusion. All the changes rapidly recovered when occlusion was released. Responses of an in-situ isolated segment on a side branch revealed conducted dilatory signals caused by the occlusions. Conclusions: Our results show that the endothelial and smooth muscle cells respond independently to arteriolar occlusion. The endothelial and smooth muscle cells do not effectively communicate in [Ca2+]i or membrane potential during acute arteriolar occlusion. Hyperpolarizing signals in endothelium cause conducted dilation.

Measurement of Membrane Potential and Intracellular Ca2+ of Arteriolar Endothelium and Smooth Muscle in Vivo

We have developed an intensity analysis technique for fluorescence microscopy that allows us to measure, in real time, the diameter and the membrane potential or intracellular calcium ([Ca2+]i) of in vivo arteriolar endothelium or smooth muscle. Cheek pouch arterioles of anesthetized hamsters were luminally or abluminally labeled with Di-8-ANEPPS, a voltage-sensitive dye, or Fura PE3, a calcium indicator. The peak fluorescence intensities of the images were used to locate the endothelium or smooth muscle. The changes in membrane potential or [Ca2+]i were determined based on the ratiometric analysis of fluorescence intensity of the endothelium or smooth muscle. Membrane depolarization of the smooth muscle using KCl caused a decrease in the ratio of emission, 620 nm/560 nm (∼6 mV/% ratio). The ratio of excitation, 340 nm/380 nm, increased with increasing free Ca2+. Methacholine, a muscarinic receptor agonist, caused arteriolar dilation (12.2 ± 0.9 μm). It produced hyperpolarization of the endothelium and smooth muscle (2.8 ± 0.6% and 2.3 ± 0.3% in ratio). Methacholine also induced an increase in [Ca2+]i (11.0 ± 1.1% in ratio) of the endothelium. In contrast, methacholine caused a biphasic change in [Ca2+]i of the smooth muscle, a rapid reduction (−3.4 ± 0.2% in ratio) followed by a prolonged increase (2.4 ± 0.2% in ratio). These results demonstrate that the peak intensity analysis can be used to determine in real time the changes in membrane potential or [Ca2+]i of in vivo endothelium or smooth muscle.

Electrophysiological Basis of Arteriolar Vasomotion in vivo

We tested the hypothesis that cyclic changes in membrane potential (E_m) underlie spontaneous vasomotion in cheek pouch arterioles of anesthetized hamsters. Diameter oscillations (∼3 min^–1) were preceded (∼3 s) by oscillations in E_m of smooth muscle cells (SMC) and endothelial cells (EC). Oscillations in E_m were resolved into six phases: (1) a period (6 ± 2 s) at the most negative E_m observed during vasomotion (–46 ± 2 mV) correlating (r = 0.87, p < 0.01) with time (8 ± 2 s) at the largest diameter observed during vasomotion (41 ± 2 µm); (2) a slow depolarization (1.8 ± 0.2 mV s^–1) with no diameter change; (3) a fast (9.1 ± 0.8 mV s^–1) depolarization (to –28 ± 2 mV) and constriction; (4) a transient partial repolarization (3–4 mV); (5) a sustained (5 ± 1 s) depolarization (–28 ± 2 mV) correlating (r = 0.78, p < 0.01) with time (3 ± 1 s) at the smallest diameter (27 ± 2 µm) during vasomotion; (6) a slow repolarization (2.5 ± 0.2 mV s^–1) and relaxation. The absolute change in E_m correlated (r = 0.60, p < 0.01) with the most negative E_m. Sodium nitroprusside or nifedipine caused sustained hyperpolarization and dilation, whereas tetraethylammonium or elevated PO₂ caused sustained depolarization and constriction. We suggest that vasomotion in vivo reflects spontaneous, cyclic changes in E_m of SMC and EC corresponding with cation fluxes across plasma membranes.

TNF-α Modulates Arteriolar Reactivity Secondary to a Change in Intimal Permeability

Inflammatory stimuli are often associated with marked changes in vascular reactivity. Tumor necrosis factor-alpha (TNF-alpha) is and important inflammatory cytokine with diverse effects including the ability to increase vascular endothelial cell permeability and to alter the structure of the endothelial cell glycocalyx. We have previously shown that arteriolar sensitivity to vasoactive materials is influenced by a barrier property of the arteriolar endothelium, and here we test the hypothesis that TNF-alpha might increase intimal permeability and thereby increase the access of circulating arginine-vasopressin (AVP) to vascular smooth muscle. Our objective in the current work was to show that TNF-alpha-mediated modulation of intimal cell permeability may produce both quantitative and qualitative alterations in vascular reactivity to arginine-vasopressin. Hamster cheek pouch arterioles (approximately 65 microm, i.d.) were double-cannulated and perfused. [Arg8]-vasopressin was applied selectively to either the luminal or the adventitial surface of isolated cannulated arterioles. The reactivity of the arterioles to vasopressin was determined in the presence and absence of TNF-alpha (0.625 microg/mL; 1 hour). Adventially applied AVP induced a concentration-dependent vasoconstriction with a threshold of approximately 1 pM, and a maximal constriction at 10 nM. In contrast, luminally applied AVP induced a biphasic response, showing a modest vasodilation in the range of 1 to 100 pM, and constrictions at doses higher than 1 nM. Maximal constrictions were not obtained with luminal doses of AVP as high as 1 microM; (i.e., at doses 100-fold higher than those that produced maximal responses with adventitial application). Dilations induced by luminal application of AVP were significantly attenuated by 10 microM Nomega-nitro-L-arginine methyl ester (L-NAME), but were not altered by 10 microM indomethacin. After treatment with TNF-alpha, the concentration-response curve for luminally applied AVP showed a more pronounced constriction and the dilator component of the agonist was eliminated. There was no change in the reactivity to adventitially applied AVP or to adventitial applications of acetylcholine. Nonspecific increases in endothelial cell permeability induced by 3-[(3-chloroamino-dopropyl)-dimethylamino]-1-propanesulfonate (CHAPS) also eliminated the potency differences between luminal and adventitial drug application. Following TNF-alpha treatment and loss of the dilator component of the response to AVP, L-NAME was still capable of reducing the arteriolar sensitivity to ACh, thus showing that the endothelial cell machinery for NO production was intact following TNF-alpha treatment. Our findings show that the reactivity of intact resistance vessels to agonists that have both endothelial-dependent and smooth muscle cell-dependent components will be complex. Reactivity is the summation of: 1) the relative sensitivities of smooth muscle and endothelial cells to AVP, 2) the release of nitric oxide or other mediators from endothelium, and 3) the restricted access of intraluminal AVP to arteriolar smooth muscle cells. Thus, cytokines, and perhaps other materials that regulate endothelial cell permeability, can modify arteriolar reactivity by altering transendothelial cell access of luminal stimuli to the smooth muscle cells, as well as by acting directly on smooth muscle or by influencing the production of endothelial cell-derived vasoactive materials. In the case of agonists that have an endothelial cell dilator component as well as a smooth muscle constrictor component, this may result in a qualitative change in response as is shown here with AVP.

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.

Constrictor responses of resistance arterioles during diabetes mellitus

While diabetes mellitus appears to alter nitric oxide synthase-dependent vasodilatation, the effect of diabetes on constrictor responses of resistance arterioles is not clear. Our goal was to examine the effect of diabetes on constrictor responses of cheek pouch arterioles. In vivo diameter of arterioles (∼50 μm) was measured in response to norepinephrine, the thromboxane analogue (U-46619) and endothelin-1 in nondiabetic and diabetic hamsters (4–6 weeks post streptozotocin). Norepinephrine (1.0 and 10 nM) and U-46619 (0.1 and 1.0 nM) produced similar dose-related vasoconstriction in nondiabetic and diabetic hamsters ( P>0.05). In contrast, vasoconstriction to endothelin-1 (0.1 and 1.0 pM) was greater in diabetic than nondiabetic hamsters ( P<0.05). Next, we examined the role of nitric oxide in basal vascular tone and whether enhanced vasoconstriction in diabetic hamsters to endothelin-1 might be related to an alteration in the modulatory role of nitric oxide. N G-monomethyl- l-arginine ( l-NMMA) (1.0, 10 and 100 μM) produced dose-related vasoconstriction in nondiabetic, but not diabetic hamsters. Further, l-NMMA did not alter vasoconstriction in response to endothelin-1 in nondiabetic and diabetic hamsters. These findings suggest that diabetes alters constriction of cheek pouch resistance arterioles to endothelin-1 which appears to be independent of the synthesis/release of nitric oxide. In addition, based upon findings using l-NMMA, it appears that there is a reduced influence of nitric oxide on basal diameter of resistance arterioles during diabetes.

Spread of vasodilatation and vasoconstriction along feed arteries and arterioles of hamster skeletal muscle.

1. In arterioles of the hamster cheek pouch, vasodilatation and vasoconstriction can spread via the conduction of electrical signals through gap junctions between cells that comprise the vessel wall. However, conduction in resistance networks supplying other tissues has received relatively little attention. In anaesthetized hamsters, we have investigated the spread of dilatation and constriction along feed arteries and arterioles of the retractor muscle, which is contiguous with the cheek pouch. 2. When released from a micropipette, acetylcholine (ACh) triggered vasodilatation that spread rapidly along feed arteries external to the muscle and arterioles within the muscle. Responses were independent of changes in wall shear rate, perivascular nerve activity, or release of nitric oxide, indicating cell-to-cell conduction. 3. Vasodilatation conducted without decrement along unbranched feed arteries, yet decayed markedly in arteriolar networks. Thus, branching of the conduction pathway dissipated the vasodilatation. 4. Noradrenaline (NA) or a depolarizing KCl stimulus evoked constriction of arterioles and feed arteries of the retractor muscle that was constrained to the vicinity of the micropipette. This behaviour contrasts sharply with the conduction of vasodilatation in these microvessels and with the conduction of vasoconstriction elicited by NA and KCl in cheek pouch arterioles. 5. Focal electrical stimulation produced constriction that spread rapidly along feed arteries and arterioles. These responses were inhibited by tetrodotoxin or prazosin, confirming the release of NA along perivascular sympathetic nerves, which are absent from arterioles studied in the cheek pouch. Thus, sympathetic nerve activity co-ordinated the contraction of smooth muscle cells as effectively as the conduction of vasodilatation co-ordinated their relaxation. 6. In the light of previous findings in the cheek pouch, the properties of vasoconstriction and vasodilatation in feed arteries and arterioles of the retractor muscle indicate that substantive differences can exist in the nature of signal transmission along microvessels of tissues that differ in structure and function.

Chronic exposure to nicotine alters endothelium-dependent arteriolar dilatation: effect of superoxide dismutase.

The first goal of this study was to determine whether chronic injection of nicotine alters endothelium-dependent arteriolar dilatation. We measured the diameter of cheek pouch resistance arterioles (approximately 50 microm in diameter) in response to endothelium-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists in control hamsters and hamsters treated with nicotine (2 microg. kg-1. day-1 for 2-3 wk). In control hamsters, acetylcholine (0.1 and 1.0 microM) dilated arterioles by 13 +/- 2 and 31 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by 18 +/- 1 and 30 +/- 1%, respectively. In contrast, acetylcholine (0.1 and 1.0 microM) dilated arterioles by only 5 +/- 2 and 12 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by only 7 +/- 2 and 13 +/- 3%, respectively, in animals treated with nicotine (P < 0.05 vs. response in control hamsters). Nitroglycerin produced similar dose-related dilatation of cheek pouch arterioles in control and nicotine-treated hamsters. Our second goal was to examine a possible mechanism for impaired endothelium-dependent arteriolar dilatation during chronic treatment with nicotine. We found that superfusion of the cheek pouch microcirculation with superoxide dismutase (150 U/ml) restored impaired endothelium-dependent, but did not alter endothelium-independent, arteriolar dilatation in hamsters treated with nicotine. Superfusion with superoxide dismutase did not alter endothelium-dependent or -independent arteriolar dilatation in control hamsters. We suggest that chronic exposure to nicotine produces selective impairment of endothelium-dependent arteriolar dilatation via a mechanism related to the synthesis/release of oxygen-derived free radicals.