A role for cyclooxygenase in aging-related changes of beta-adrenoceptor-mediated relaxation in rat aortas.

beta-Adrenoceptor-mediated vasorelaxation decreases with age in various vascular beds. The present study investigated the roles of cyclooxygenase (COX) on beta-adrenoceptor vasorelaxation by isoprenaline in 8- and 54-week-old rat aortas. The vasorelaxation responses by isoprenaline (0.03-3 microM) were significantly reduced in 54-week-old aortas compared to 8-week. Addition of the non-selective COX inhibitors indomethacin (10 microM) or aspirin (10 microM) restored isoprenaline vasorelaxation of 54-week-old aortas to levels found in 8-week-old aortas. This suggests the involvement of COX prostanoids in the age-related reduction of beta-adrenoceptor vasorelaxation. Immunohistochemistry revealed greater levels of COX-1 and COX-2 staining in 54-week-old aortas compared to 8-week with expression located mainly in medial smooth muscle. An age-linked increase in COX-1 and COX-2 protein was found in cremaster arterioles of 54-week-old rats (compared to 8-week) mainly in the endothelial layer. The age-related increase in COX-1 and COX-2 protein led to elevation of prostacyclin (measured as 6-keto prostaglandin F(1alpha)) and thromboxane A(2) (measured as thromboxane B(2)) in 54-week compared to 8-week-old aortas. Endothelium removal in 54-week aortas markedly reduced the 6-keto prostaglandin F(1alpha) level, thus suggesting an endothelial source for elevated prostacyclin. These findings in combination with the effects of COX inhibitors suggest that the age-related decrease in beta-adrenoceptor vasorelaxation by isoprenaline is due to an age-linked increase in COX expression, which elevates production of COX-derived vasoactive prostanoids.

Intracoronary basic fibroblast growth factor enhances myocardial collateral perfusion in dogs.

OBJECTIVE: In preparation for clinical trials of basic fibroblast growth factor (bFGF) to treat ischemic heart disease, we sought to identify a clinically feasible method of bFGF administration. BACKGROUND: Basic FGF has been shown to promote collateral development after experimentally induced coronary occlusion; however, methods of bFGF delivery that have been shown to be effective in previous investigations would not be practical for clinical use. METHODS: Four randomized, blinded, controlled investigations were conducted independently and sequentially in an established canine model. For all studies, dogs underwent operative placement of proximal left circumflex coronary artery ameroid constrictors. The four investigational regimens included: 1) bFGF by central venous bolus injection, 1,740 microg/day for one, two or seven days; 2) bFGF by intravenous infusion, 100 microg/kg body weight per day for seven days; 3) bFGF by pericardial instillation, 2,000 microg/day for 7 days; and 4) bFGF by intracoronary injection (Judkin's technique), 100 microg/kg per day for one or two days. Each substudy included a contemporaneous vehicle control group. Collateral perfusion (microspheres) was assessed during maximal coronary vasodilation during the first month after ameroid placement. RESULTS: Maximal collateral perfusion in dogs that received intracoronary bFGF for two days exceeded that of concurrent control dogs by 31% (p < 0.01). Perfusion was not increased in dogs that received single-dose intracoronary bFGF. Basic FGF administration by central venous bolus injection, intravenous infusion and pericardial injection failed to enhance collateral perfusion. CONCLUSIONS: Administration of bFGF by the intracoronary route, an intervention that is feasible in patients, augments collateral development in dogs. These data provide a rationale for clinical testing of intracoronary bFGF in ischemic heart disease.

Effects of reduced calcium ion concentration and of diltiazem on vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in rat isolated tail artery.

In isolated, perfused proximal segments of Sprague-Dawley rat tail artery, idazoxan (100 nmol l-1) displaced the concentration-response curve to noradrenaline (NA) to the right. The log shift of the NA concentration-response curve was greater at lower concentrations than at higher concentrations of NA. Idazoxan (100 nmol l-1) had no effect on responses to electrical stimulation. Prazosin (10 nmol l-1) displaced the concentration-response curve to NA to the right as well as markedly reducing responses to sympathetic nerve stimulation. The concentration-response curve to NA, obtained after reducing the concentration of calcium ions in the Krebs solution from 2.5 to 0.6 mmol l-1, was significantly displaced to the right. Responses to sympathetic nerve stimulation were not affected by this reduction in the concentration of calcium ions. Diltiazem (1 and 10 mumol l-1) significantly displaced the concentration-response curve to NA to the right but had no effect on sympathetic nerve stimulation. These in vitro results in peripheral arterial smooth muscle confirm the findings of previous in vivo studies which suggest that alpha2-adrenoceptors contribute to the vasoconstrictor responses elicited by alpha-adrenoceptor agonists and that these responses but not those mediated by alpha1-adrenoceptors are dependent on extracellular calcium.

Differential effects of hydroxocobalamin on NO-mediated relaxations in rat aorta and anococcygeus muscle.

In rat aortic rings, hydroxocobalamin (10-30 microM) produced concentration-dependent reductions of the relaxant action of nitric oxide (NO) and the endothelium-dependent, NO-mediated, relaxant action of acetylcholine. In anococcygeus muscles, hydroxocobalamin (10-30 microM) reduced but also prolonged, NO-induced relaxations, but had no effect on non-adrenergic, non-cholinergic-mediated relaxations. Hydroxocobalamin had no effect on the NO-independent relaxant action of papaverine in either tissue. It is suggested that hydroxocobalamin sequesters NO by forming nitrosocobalamin. Nitrosocobalamin did not relax aortic rings, but produced a slowly developing and prolonged relaxation of anococcygeus muscles.

Vasoconstrictor responses of rat tail artery to sympathetic nerve stimulation contain a component due to activation of postjunctional beta- or alpha 2-adrenoceptors.

The role of alpha 1-, alpha 2- and beta-adrenoceptors in vasoconstrictor responses to sympathetic nerve stimulation was investigated in perfused proximal segments of rat tail artery by using selective blocking drugs. Prazosin (1 nM) markedly reduced the responses but idazoxan (100 nM) did not, and propranolol (1 microM) significantly enhanced them, indicating that the vasoconstriction was due to activation of alpha 1-adrenoceptors and that it was partly counteracted by a vasodilator component due to activation of beta-adrenoceptors. In the presence of propranolol, idazoxan or reduction of the concentration of Ca2+ in the perfusing solution from 2.5 to 0.63 mM significantly reduced responses to sympathetic nerve stimulation, indicating that a component of the vasoconstrictor response was due to activation of alpha 2-adrenoceptors. Forskolin, which increases cyclic AMP levels independently of beta-adrenoceptors, reduced responses to sympathetic nerve stimulation to a greater extent in the presence than in the absence of propranolol and this effect was additive with that of prazosin but not idazoxan. It is concluded that activation of beta-adrenoceptors inhibits the component of responses to sympathetic nerve stimulation due to activation of alpha 2-adrenoceptors because of an inhibitory effect of cyclic AMP on Ca2+ channels linked to alpha 2-adrenoceptors.

Greater activation of smooth muscle alpha-2 adrenoceptors by epinephrine in distal than in proximal segments of rat tail artery.

The effects of selective blockade of alpha-1 and alpha-2 adrenoceptors on the vasoconstrictor responses to epinephrine (EPI) and norepinephrine (NOR) were compared in perfused/superfused proximal and distal segments of rat tail artery. The influence of neuronal uptake and of activation of beta adrenoceptors was also investigated. EPI was more potent in distal than in proximal segments. The antagonistic effect of idazoxan (100 nM) against EPI was greater in distal than in proximal segments, whereas the opposite result was obtained with prazosin (0.1-10 nM). No such difference were observed when NOR was used as agonist. Reducing the calcium ion concentration had a greater inhibitory effect on EPI in distal than in proximal segments. Cocaine (4 microM) increased responses to EPI and NOR to a greater extent in proximal than in distal segments. In the presence of cocaine, in proximal segments, antagonism of EPI by prazosin was reduced, whereas in distal segments, antagonism by idazoxan and the inhibitory effect of a reduction in calcium ion concentration were reduced. Propranolol (1 microM) increased responses to EPI and NOR to a greater extent in proximal than in distal segments. In the presence of propranolol, antagonism of EPI by both prazosin and idazoxan was reduced in proximal segments, and the inhibitory effect of a reduction in calcium ion concentration was lost. Forskolin (1 microM) inhibited responses to EPI and prevented the antagonistic effect of idazoxan, but not that of prazosin. From the results obtained it is suggested that smooth muscle alpha-2 adrenoceptors are distributed differently in the proximal and distal ends of the rat tail artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential activation of adrenoceptor subtypes by noradrenaline applied from the intimal or adventitial surfaces of rat isolated tail artery.

1. The vasoconstrictor effects of noradrenaline applied to the intimal and adventitial surfaces of perfused segments of rat tail artery in the presence and absence of endothelium were studied. 2. Noradrenaline was about six times more potent as a vasoconstrictor when applied to the intimal than to the adventitial surface. Cocaine (25 mumol/L) enhanced responses to adventitial noradrenaline to a greater extent than those to intimal noradrenaline. A high concentration of propranolol (1 mumol/L) had a similar effect. 3. The vasoconstriction elicited by adventitial noradrenaline declined from a peak whereas that to intimal noradrenaline remained steady. A low concentration of propranolol (0.1 mumol/L) abolished the decline in the response to adventitial noradrenaline. 4. The alpha 1- and alpha 2-adrenoceptor antagonists prazosin (1 nmol/L) and idazoxan (100 nmol/L) significantly reduced responses to intimal and adventitial noradrenaline in the presence or absence of endothelium. 5. Removal of endothelium enhanced responses to intimal but not adventitial noradrenaline. Idazoxan produced a significantly greater reduction of responses to noradrenaline in the absence than in the presence of endothelium, and was more effective against intimal than adventitial noradrenaline. Similar effects were produced by the nitric oxide synthase inhibitor L-NAME (30 mumol/L). 6. It was concluded that noradrenaline acts on both alpha 1- and alpha 2-adrenoceptors to produce vasoconstriction: the alpha 1-adrenoceptors appear to be uniformly distributed, whereas alpha 2-adrenoceptors are located nearer the intima. Intimal noradrenaline also acts on endothelial alpha 2-adrenoceptors to release EDRF which counteracts the vasoconstrictor action of noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitation of noradrenaline release by isoprenaline is not mediated by angiotensin II in mouse atria and rat tail artery.

The hypothesis that facilitation of noradrenaline release by prejunctional beta-adrenoceptors is linked to the local generation of angiotensin II was investigated in rat tail artery and mouse atria which were incubated with [3H]-noradrenaline. The outflow of radioactivity induced by electrical field stimulation of the tissues was taken as an index of noradrenaline release. In mouse atria, angiotensin II (0.01 microM) enhanced the stimulation-induced (S-I) noradrenaline release and this was blocked by saralasin (0.1 microM). Isoprenaline (0.01 microM) also enhanced the S-I release of noradrenaline, but this was not blocked by either saralasin (0.1 microM and 0.3 microM) or captopril (5 microM). Furthermore, the facilitatory effects of angiotensin II (0.1 microM) and isoprenaline (0.1 microM) were additive. In rat tail artery, the facilitatory effect of angiotensin II (0.01 microM) on noradrenaline release was blocked by saralasin (0.1 microM). The facilitatory effect of isoprenaline (0.1 microM) was blocked by propranolol (1 microM) but not by saralasin (0.1 microM) nor captopril (5 microM). These results indicate that beta-adrenoceptor-mediated facilitation of noradrenaline release occurs independently of angiotensin II in mouse atria and rat tail artery.

Myogenic reactivity of rat epineurial arterioles: potential role in local vasoregulatory events.

Local control of neural blood flow is considered to reside in innervation of epineurial and endoneurial arterioles rather than in intrinsic autoregulatory mechanisms. With the use of an isolated vessel preparation and an in vivo approach, the present studies examined intrinsic vasomotor responsiveness of epineurial arterioles. Segments of epineurial arterioles, cannulated on glass micropipettes (40 micrometers) and pressurized in the absence of intraluminal flow, showed sustained pressure-dependent (30-90 mmHg) vasoconstriction and acute myogenic reactivity. Myogenic tone was unaffected by phentolamine (10(-6) M). Removal of extracellular Ca(2+) resulted in loss of spontaneous tone and passive behavior. Concentration-response curves for norepinephrine (10(-9)-3 x 10(-6) M) and relaxation to both acetylcholine (10(-8)-10(-5) M) and adenosine (10(-8)-10(-4) M) were obtained. Acetylcholine dilator responses were inhibited by N(G)-nitro-L-arginine methyl ester. Epineurial blood flow was measured in vivo using a laser-Doppler flow probe. Blood flow declined over a 2-h period after surgery, and during this time preparations developed responsiveness to the dilator acetylcholine. Phentolamine blocked vasoconstrictor responses to exogenous norepinephrine but only partially reversed the in vivo baseline tone. The time-dependent decline in epineurial blood flow was observed despite the presence of tetrodotoxin (1 microM), further confirming that tone was predominantly caused by myogenic rather than neurogenic mechanisms. It is concluded that because epineurial arterioles exhibit intrinsic myogenic reactivity, they have the potential to participate in local regulation of neural hemodynamics independently of their own innervation.

Matrix protein glycation impairs agonist-induced intracellular Ca2+ signaling in endothelial cells.

Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress.

Approaches for introducing peptides into intact and functional arteriolar smooth muscle: manipulation of protein kinase-based signalling.

1. An exact understanding of signal transduction pathways within intact and functional arteriolar smooth muscle is made difficult by limited access to the intracellular environment due to the cell membrane. The aim of the present studies was to determine the feasibility of using polycationic lipids and reverse permeabilization for the introduction of peptide inhibitors into smooth muscle cells of the intact arteriolar wall. 2. Isolated cannulated arterioles were exposed to polycationic lipid preparations together with varying concentrations of the protein beta-galactosidase (30-90 microg/mL). Similar experiments were also performed using cultured smooth muscle cells. Staining for the chromogenic substrate of beta-galactosidase (5-bromo-4-chloro-3-indolyl-beta-d-galactosidase; X-gal) demonstrated incorporation of the protein into cultured cells but not intact arteriolar smooth muscle. Similarly, polycationic lipid treatment did not enable loading of arteriolar smooth muscle (as assessed by cAMP-mediated vasodilation) with the protein kinase (PK) A inhibitory peptide PKI. 3. In contrast, reverse permeabilization, using high ATP concentrations in the presence of EGTA enabled introduction of PKI and inhibition of forskolin-mediated vasodilatation. Furthermore, arterioles maintained full viability following reverse permeabilization, as demonstrated by an ability to develop spontaneous myogenic tone. 4. Reverse permeabilization provides a method for introducing peptide inhibitors into functional arteriolar smooth muscle and manipulating signal transduction. Protein transfection using polycationic lipids appears to be limited by the barrier provided by the adventitia or inherent differences between cells under cultured conditions compared within the intact arteriole.