Pieter Sipkema

Physiological concentrations of insulin induce endothelin-mediated vasoconstriction during inhibition of NOS or PI3-kinase in skeletal muscle arterioles

OBJECTIVE To determine the roles of nitric oxide, endothelin-1 and phosphatidylinositol 3-kinase (PI3-kinase) in acute responses of isolated rat skeletal muscle arterioles to insulin. METHODS Rat cremaster first order arterioles were separated from surrounding tissue, cannulated in a pressure myograph and responses to insulin (4 microU/ml-3.4 mU/ml) were studied without intraluminal blood or flow. RESULTS Insulin alone did not significantly affect arteriolar diameter. Non-selective antagonism of endothelin receptors, with PD-142893, uncovered insulin-induced vasodilatation (25+/-8% from baseline at 3.4 mU/ml), which was abolished by inhibition of NO synthesis with N(G)-nitro-L-arginine (L-NA). Inhibition of NO synthesis alone uncovered insulin-induced vasoconstriction at physiological concentrations (21+/-5% from baseline diameter at 34 microU/ml), which was abolished by PD-142893. The NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) inhibited insulin-induced vasoconstriction during NOS inhibition, even at a concentration that did not elicit vasodilatation itself. Inhibition of PI3-kinase, an intracellular mediator of insulin-induced NO production, with wortmannin, also uncovered insulin-induced vasoconstriction (13+/-3% from baseline at 34 microU/ml) that was abolished by PD-142893. CONCLUSIONS Insulin induces both nitric oxide and endothelin-1 activity in rat cremaster first-order arterioles. This study demonstrates for the first time that vasoconstrictive effects of physiological concentrations of insulin during inhibition of NOS activity are mediated by endothelin and that insulin induces endothelin-1-mediated vasoconstriction in isolated skeletal muscle arterioles during inhibition of PI3-kinase. These findings support the hypothesis of altered microvascular reactivity to insulin in conditions of diminished PI3-kinase activity, a prominent feature of insulin resistance.

Physiological Concentrations of Insulin Induce Endothelin-Dependent Vasoconstriction of Skeletal Muscle Resistance Arteries in the Presence of Tumor Necrosis Factor-α Dependence on c-Jun N-Terminal Kinase

Objective—Tumor necrosis factor-α (TNF-α) has been linked to obesity-related insulin resistance and impaired endothelium-dependent vasodilatation, but the mechanisms have not been elucidated. To investigate whether TNF-α directly impairs insulin-mediated vasoreactivity in skeletal muscle resistance arteries and the role of c-Jun N-terminal kinase (JNK) in this interference. Methods and Results—Insulin-mediated vasoreactivity of isolated resistance arteries of the rat cremaster muscle to insulin (4 to 3400 &mgr;U/mL) was studied in the absence and presence of TNF-α (10 ng/mL). Although insulin or TNF-α alone did not affect arterial diameter, insulin induced dose-dependent vasoconstriction of cremaster resistance arteries in the presence of TNF-α, (−12±1% at 272 &mgr;U/mL). Blocking endothelin receptors in the absence of TNF-α uncovered insulin-mediated vasodilatation (18±6% at 272 &mgr;U/mL) but not in the presence of TNF-α (2±2% at 272 &mgr;U/mL), showing that TNF-α inhibits vasodilator effects of insulin. Using digital imaging microscopy, we discovered that TNF-α activates JNK in arterial endothelium, visible as an increase in phosphorylated JNK. Moreover, inhibition of JNK with the cell-permeable peptide inhibitor L-JNKI abolished insulin-mediated vasoconstriction in the presence of TNF-α, showing that JNK is required for interaction between TNF-α and insulin. Conclusions—TNF-α inhibits vasodilator but not vasoconstrictor effects of insulin in skeletal muscle resistance arteries, resulting in insulin-mediated vasoconstriction in the presence of TNF-α. This effect of TNF-α is critically dependent on TNF-α–mediated activation of JNK.

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC(50))) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching -5.15+/-0.79 and before stretching -6.71+/-0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.

Characterization of the Arterial System in the Time Domain

The impulse response function and the input impedance of the systemic arterial tree emphasize different aspects of this system. The impulse response function is calculated via inverse Fourier transformation of the input impedance. The effects of truncation of the impedance are reduced by subjecting the data to a Dolph-Chebyshev filter. The impulse response functions of a windkessel model, a uniform tube model, and of the arterial system of the dog, are given. The impulse response functions of the windkessel model and of the arterial system of the control dog show a sharp initial peak followed by an exponential decay (equal decay time as that of the diastolic pressure tracing). The height of the decay extrapolated to time zero is related to total arterial compliance. Total arterial compliance calculated in this way agrees with the value calculated from the ratio of the time constant of the diastolic pressure decay and peripheral resistance. The presence of peaks in the impulse response function indicates a distinct reflection site as shown in the uniform tube model and found in the dog with balloon occlusion of the descending aorta. The measurement of the time intervals between these peaks and the start of excitation together with the pulse wave velocity enable us to calculate the distance between the location of the reflecting site and the heart.

Activation of AMP-Activated Protein Kinase by 5-Aminoimidazole-4-Carboxamide-1-β-d-Ribofuranoside in the Muscle Microcirculation Increases Nitric Oxide Synthesis and Microvascular Perfusion

Objective—To investigate the effects of activation of the AMP-activated protein kinase (AMPK) on muscle perfusion and to elucidate the mechanisms involved. Methods and Results—In a combined approach, we studied the vasoactive actions of AMPK activator by 5-aminoimidazole-4-carboxamide-1-&bgr;-d-ribofuranoside (AICAR) on rat cremaster muscle resistance arteries (≈100 &mgr;m) ex vivo and on microvascular perfusion in the rat hindlimb in vivo. In isolated resistance arteries, AICAR increased Thr172 phosphorylation of AMPK in arteriolar endothelium, which was predominantly located in microvascular endothelium. AICAR induced vasodilation (19±4% at 2 mmol/L, P<0.01), which was abolished by endothelium removal, inhibition of NO synthase (with N-nitro-l-arginine), or AMPK (with compound C). Smooth muscle sensitivity to NO, determined by studying the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP), was not affected by AICAR except at the highest dose. AICAR increased endothelial nitric oxide synthase activity, as indicated by Ser1177 phosphorylation. In vivo, infusion of AICAR markedly increased muscle microvascular blood volume (≈60%, P<0.05), as was evidenced by contrast-enhanced ultrasound, without effects on blood pressure, femoral blood flow, or hind leg glucose uptake. Conclusion—Activation of AMPK by AICAR activates endothelial nitric oxide synthase in arteriolar endothelium by increasing its Ser1177 phosphorylation, which leads to vasodilation of resistance arteries and recruitment of microvascular perfusion in muscle.

Protein Kinase C θ Activation Induces Insulin-Mediated Constriction of Muscle Resistance Arteries

OBJECTIVE—Protein kinase C (PKC) θ activation is associated with insulin resistance and obesity, but the underlying mechanisms have not been fully elucidated. Impairment of insulin-mediated vasoreactivity in muscle contributes to insulin resistance, but it is unknown whether PKCθ is involved. In this study, we investigated whether PKCθ activation impairs insulin-mediated vasoreactivity and insulin signaling in muscle resistance arteries. RESEARCH DESIGN AND METHODS—Vasoreactivity of isolated resistance arteries of mouse gracilis muscles to insulin (0.02–20 nmol/l) was studied in a pressure myograph with or without PKCθ activation by palmitic acid (PA) (100 μmol/l). RESULTS—In the absence of PKCθ activation, insulin did not alter arterial diameter, which was caused by a balance of nitric oxide–dependent vasodilator and endothelin-dependent vasoconstrictor effects. Using three-dimensional microscopy and Western blotting of muscle resistance arteries, we found that PKCθ is abundantly expressed in endothelium of muscle resistance arteries of both mice and humans and is activated by pathophysiological levels of PA, as indicated by phosphorylation at Thr538 in mouse resistance arteries. In the presence of PA, insulin induced vasoconstriction (21 ± 6% at 2 nmol/l insulin), which was abolished by pharmacological or genetic inactivation of PKCθ. Analysis of intracellular signaling in muscle resistance arteries showed that PKCθ activation reduced insulin-mediated Akt phosphorylation (Ser473) and increased extracellular signal–related kinase (ERK) 1/2 phosphorylation. Inhibition of PKCθ restored insulin-mediated vasoreactivity and insulin-mediated activation of Akt and ERK1/2 in the presence of PA. CONCLUSIONS—PKCθ activation induces insulin-mediated vasoconstriction by inhibition of Akt and stimulation of ERK1/2 in muscle resistance arteries. This provides a new mechanism linking PKCθ activation to insulin resistance.

Signal transduction in spontaneous myogenic tone in isolated arterioles from rat skeletal muscle

OBJECTIVE The mechanism of spontaneous myogenic tone was investigated in isolated arteriolar segments. METHODS Arterioles were isolated from rat cremaster muscle. Segments were endothelium-denuded and mounted in a pressure myograph at 75 mmHg. Under this condition, segments spontaneously constricted from a passive diameter of 167 +/- 3 to 82 +/- 4 microns (n = 41). The effects of several inhibitors were tested on the maintenance of myogenic tone. RESULTS Gadolinium (10(-6)-10(-4) M), a putative inhibitor of stretch-activated cation channels, was ineffective. The phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) induced a dose-dependent inhibition of tone. NCDC inhibited phenylephrine- (10(-6) M), but not potassium buffer-induced (100 mM) constriction. The protein kinase C (PKC) inhibitors staurosporine, chelerythrine and calphostin C inhibited myogenic tone in a concentration-dependent manner. At an intermediate concentration, calphostin C selectively inhibited phenylephrine-induced constriction. However, all PKC inhibitors abolished responses to phenylephrine and potassium buffer at higher concentrations. The cytochrome P450 inhibitor 17-ODYA (0.3-3 x 10(-6) M) did not inhibit myogenic tone. CONCLUSIONS No evidence was found for a role of gadolinium-sensitive, stretch-activated cation channels or cytochrome P450 metabolites. On the other hand, both PLC and PKC contribute to the maintenance of myogenic tone.

Components of acetylcholine-induced dilation in isolated rat arterioles

Acetylcholine-induced dilation was studied in cannulated resistance arteries of rat cremaster muscle. Pressurized arteriolar segments (internal diameter: 175 +/- 2 microm) developed spontaneous tone (90 +/- 2 microm). Application of acetylcholine (0.1 and 0.3 microM) resulted in a transient dilation followed by a steady-state dilatory response. In the presence of N(G)-nitro-L-arginine (L-NNA) approximately 70% of the transient dilation was resistant to nitric oxide inhibition, whereas the steady-state response was abolished. Further experiments using 0.1 microM acetylcholine (no L-NNA present) were aimed to inhibit synthesis or action of the mediator of the transient component (amplitude: 39 +/- 2.8 microm). A high-potassium buffer (30-50 mM) abolished this transient dilation (1.3 +/- 1.3 microm), suggesting that the dilation is mediated by an endothelium-derived hyperpolarizing factor (EDHF). This putative EDHF-mediated dilation is strongly reduced by cytochrome P-450 inhibitors miconazole (11 +/- 1.3 microm) and SKF-525a (4.8 +/- 4.5 microm). The transient component is inhibited by tetraethylammonium but not by glibenclamide, indicating it is mediated by opening of Ca2+-activated K+ channels. Interestingly, inhibition of the transient component was followed by a subsequent decrease of the nitric oxide-mediated part of the response to acetylcholine. Thus a transient dilation, mediated by a cytochrome P-450 metabolite, precedes and possibly stimulates nitric oxide-mediated dilation in acetylcholine-induced dilation.Acetylcholine-induced dilation was studied in cannulated resistance arteries of rat cremaster muscle. Pressurized arteriolar segments (internal diameter: 175 ± 2 μm) developed spontaneous tone (90 ± 2 μm). Application of acetylcholine (0.1 and 0.3 μM) resulted in a transient dilation followed by a steady-state dilatory response. In the presence of N G-nitro-l-arginine (l-NNA) ∼70% of the transient dilation was resistant to nitric oxide inhibition, whereas the steady-state response was abolished. Further experiments using 0.1 μM acetylcholine (no l-NNA present) were aimed to inhibit synthesis or action of the mediator of the transient component (amplitude: 39 ± 2.8 μm). A high-potassium buffer (30-50 mM) abolished this transient dilation (1.3 ± 1.3 μm), suggesting that the dilation is mediated by an endothelium-derived hyperpolarizing factor (EDHF). This putative EDHF-mediated dilation is strongly reduced by cytochrome P-450 inhibitors miconazole (11 ± 1.3 μm) and SKF-525a (4.8 ± 4.5 μm). The transient component is inhibited by tetraethylammonium but not by glibenclamide, indicating it is mediated by opening of Ca2+-activated K+ channels. Interestingly, inhibition of the transient component was followed by a subsequent decrease of the nitric oxide-mediated part of the response to acetylcholine. Thus a transient dilation, mediated by a cytochrome P-450 metabolite, precedes and possibly stimulates nitric oxide-mediated dilation in acetylcholine-induced dilation.

Identification of canine coronary resistance and intramyocardial compliance on the basis of the waterfall model

This study was performed to elucidate the effects of cardiac contraction on coronary pressure-flow relations. On the basis of the waterfall mechanism, a lumped model of the coronary arterial system is presented consisting of a proximal (epicardial) compliance, a coronary resistance, and an intramyocardial compliance. A “back”-pressure, assumed to be proportional (constant k) to left ventricular pressure, impedes flow. From steady-state measurements of circumflex coronary artery flow and inflow pressure, together with left ventricular pressure, the values of the three model parameters and the constant k have been estimated. In the control condition proximal compliance is found to be 1.7×10−12 m4s2kg−1, intramyocardial compliance 110×10−12m4s2kg−1, and resistance 7.5×109kgm−4s−1. The proportionality constant k is close to unity. Effects of changes in left ventricular pressure and inflow pressure and the effect of vasoactive drugs on the parameters are also investigated. Changes in coronary resistance are always opposite to changes in intramyocardial compliance. Sensitivity analysis showed that epicardial compliance plays its major role during isovolumic contraction and relaxation; resistance plays a role throughout the cardiac cycle but is more important in diastole than in systole, whereas intramyocardial compliance plays a role in systole and in early diastole.

Alpha-1-Adrenoceptor Stimulation Induces Nitric Oxide Release in Rat Pulmonary Arteries

Adrenergic stimulation is often used to induce tone in clinical and intact animal studies and in isolated vessel segments. In some studies it has been found that a nitric oxide synthase (NOS) inhibitor or removal of the vascular endothelium augments the adrenergic-mediated vasoconstriction suggesting that endothelium-derived nitric oxide (NO) plays a role in adrenergic vasoconstriction [1, 2]. The aim of the present study was to investigate whether stimulation of ·1-adrenoceptors induces NO release in rat pulmonary arteries. We confirmed that inhibition of NOS in isolated pulmonary arteries results in an increased phenylephrine-mediated vasoconstriction. We also found that phenylephrine induces NO release in pulmonary vessels as detected with an NO sensor and that ·1-adrenoceptor blockade abolishes this NO release.