Mechanism of action of angiotensin II in human isolated subcutaneous resistance arteries.

1. Human isolated subcutaneous arteries were mounted in a myograph and isometric tension measured. In some experiments, intracellular calcium [Ca(2+)]i was also measured using fura-2. 2. Angiotensin II (100 pM - 1 microM) increased [Ca(2+)]i and tone in a concentration-dependent manner. The effects of angiotensin II (100 nM) were inhibited by an AT1-receptor antagonist, candesartan (100 pM). 3. Ryanodine (10 microM), had no effect on angiotensin II-induced responses, but removal of extracellular Ca(2+) abolished angiotensin II-induced rise in [Ca(2+)]i and tone. Inhibition of Ca(2+) entry by Ni(2+) (2 mM), also inhibited angiotensin II responses. The dihydropyridine, L-type calcium channel antagonist, amlodipine (10 microM), only partially attenuated angiotensin II responses. 4. Inhibition of protein kinase C (PKC) by chelerythrine (1 microM), or by overnight exposure to a phorbol ester (PDBu; 500 nM) had no effect on angiotensin II-induced contraction. 5. Genistein (10 microM), a tyrosine kinase inhibitor, inhibited angiotensin II-induced contraction, but did not inhibit the rise in [Ca(2+)]i, suggesting that at this concentration it affected the calcium sensitivity of the contractile apparatus. Genistein did not affect responses to norepinephrine (NE) or high potassium (KPSS). 6. A selective MEK inhibitor, PD98059 (30 microM), inhibited both the angiotensin II-induced contraction and rise in [Ca(2+)]i, but had no effect on responses to NE or KPSS. 7. AT1 activation causes Ca(2+) influx via L-type calcium channels and a dihydropyridine-insensitive route, but does not release Ca(2+) from intracellular sites. Activation of tyrosine kinase(s) and the ERK 1/2 pathway, but not classical or novel PKC, also play a role in angiotensin II-induced contraction in human subcutaneous resistance arteries.

Action of AT1 receptor antagonists on angiotensin II-induced tone in human isolated subcutaneous resistance arteries.

1. Human isolated subcutaneous arteries were studied under isometric conditions in a myograph. 2. Addition of angiotensin II (AII) induced a concentration-dependent increase in tone in isolated arteries. The active metabolite of candesartan (CV 11974), losartan and the active metabolite of losartan, E-3174 antagonized AII-induced tone in a non-competitive manner, but the AT2 selective antagonist, PD123319, was without effect on responses to AII. The effects of candesartan, losartan and E-3174 were analysed using a classical model of non-competitive antagonism and a two-state receptor model. 3. Mechanical removal of the endothelium; pre-incubation with Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME); pre-incubation with indomethacin, a cyclo-oxygenase inhibitor; or pre-incubation with BQ 485, an endothelin antagonist; had no significant effect on contractions induced by AII. 4. Our results suggest AII contracts human isolated resistance arteries by an action on AT1 receptors and does not involve release of endothelial factors. Use of a two-state receptor model successfully described the action of the AT1 antagonists without sacrificing assumptions regarding the competitive nature of binding of these antagonists.

Inhibition of carbonic anhydrase accounts for the direct vascular effects of hydrochlorothiazide.

Hydrochlorothiazide has been shown to exert direct vasodilator effects by activation of calcium-activated potassium (KCa) channels in human and guinea pig isolated resistance arteries. Since hydrochlorothiazide binds to and inhibits the enzyme carbonic anhydrase and because KCa channel activation is pH sensitive, we investigated the role of intracellular and extracellular carbonic anhydrase in the vascular effects of thiazide diuretics. Small arteries were isolated from guinea pig mesentery and studied by use of a microvascular myograph technique. In some experiments, tone and intracellular pH (pHi) were measured simultaneously with 2', 7'-bis(2-carboxyethyl)-5(6)'-carboxyfluorescein (BCECF-AM). Bendroflumethiazide, a thiazide diuretic with minimal inhibitory effects on carbonic anhydrase, had little effect on noradrenaline-induced tone (16+/-8% relaxation) compared with hydrochlorothiazide (74+/-12% relaxation). In contrast to hydrochlorothiazide, the action of bendroflumethiazide was unaffected by 100 nmol/L charybdotoxin, a selective blocker of KCa channels. All inhibitors of carbonic anhydrase relaxed noradrenaline-induced tone in a concentration-dependent manner, and this effect was blocked by charybdotoxin. Hydrochlorothiazide and the inhibitors of carbonic anhydrase failed to relax tone induced by a depolarizing potassium solution. Acetazolamide and hydrochlorothiazide increased pHi by 0.27+/-0.07 and 0.21+/-0.04, respectively, whereas bendroflumethiazide had a much smaller effect: 0.06+/-0.03. The rise in pHi induced by any agent was not inhibited by charybdotoxin. The vasorelaxant effect of hydrochlorothiazide is shared by other inhibitors of carbonic anhydrase. Inhibitors of carbonic anhydrase, but not bendroflumethiazide, cause intracellular alkalinization, which is associated with KCa channel opening. These data suggest that the vasodilator effect of thiazide diuretics results primarily from inhibition of vascular smooth muscle cell carbonic anhydrase, which results in a rise in pHI, leading to KCa channel activation and vasorelaxation.

Action of ryanodine on neurogenic responses in rat isolated mesenteric small arteries.

1. Rat mesenteric (approximately 250 microns) were set up in a single-channel isometric myograph designed to allow with 6 microM fura-2AM for 2 h and simultaneous recordings of neurogenic contraction (force) and intracellular calcium [Ca2+]i were obtained. In other experiments, arteries were loaded with 1 microCi ml-1 [3H]-noradrenaline (NA) for 30 min in order to measure release of [3H]-NA in response to field stimulation to examine whether ryanodine directly inhibited neuronal release of NA. 2. Arteries were activated by single intermittent field stimulation or continuously to excite intrinsic sympathetic nerves, or by cumulative addition of noradrenaline (1 nM-10 microM) to the bathing solution. 3. Pre-incubation with ryanodine markedly inhibited the contraction and [Ca2+]i release in response to single-pulse nerve stimulation. Ryanodine also inhibited an early phasic component of the response to continuous field stimulation and reduced the rate of rise in force in response to continuous field stimulation. However, stable maximal contraction and [Ca2+]i in response to continuous field stimulation as well as maximal responses to exogenous NA were unaffected. Release of [3H]-NA in response to single intermittent field stimulation was not affected by ryanodine when compared to vehicle. 4. Our results suggest that brief intermittent activation of intramural sympathetic nerves increases [Ca2+]i and contracts small arteries primarily by releasing Ca2+ from a ryanodine-sensitive intracellular store. In contrast, the stable rise in tone and [Ca2+]i resulting from continuous nerve stimulation may largely depend on sources of Ca2+ other than the ryanodine-sensitive intracellular store.

Inhibition of norepinephrine and caffeine-induced activation by ryanodine and thapsigargin in rat mesenteric arteries.

We examined the effects of ryanodine and thapsigargin on changes in cytoplasmic [Ca2+] (Cai) and muscle tension in rat mesenteric resistance arteries induced by norepinephrine (NE) and caffeine. Both ryanodine and thapsigargin markedly inhibited the increase in Cai and contractile responses to caffeine in physiological saline and to NE and caffeine in calcium-free conditions. In contrast, peak responses to potassium depolarisation and NE in physiological saline appeared little affected, although time taken to achieve 50% of peak response after addition of NE was slowed after ryanodine and thapsigargin treatment. Neither ryanodine nor thapsigargin altered resting tone or Cai or the Ca2+ sensitivity of contraction under depolarized conditions. The NE concentration-response relationship was not significantly altered after ryanodine or thapsigargin. Ryanodine and thapsigargin inhibit the release of intracellular Ca2+ stores by NE and caffeine. Inhibition of release of intracellular Ca2+ by NE has only slight effects on contractile responses of mesenteric resistance arteries when extracellular Ca2+ is present.

Action of heparin and ruthenium red on responses of reversibly-permeabilised rat mesenteric arteries.

Heparin and ruthenium red were introduced intracellularly into rat mesenteric resistance arteries via reversible-permeabilisation. Heparin and ruthenium red inhibited contractile responses to noradrenaline, but not caffeine in Ca(2+)-free conditions. Neither heparin nor ruthenium red significantly inhibited peak contractile responses to K+, noradrenaline or caffeine in physiological saline, although heparin significantly increased the time taken for peak force to develop in response to noradrenaline. Noradrenaline and calcium concentration-response relationships were unaffected by heparin. Experiments with permeabilised, fura-2 loaded vessels indicated that heparin inhibited Ca2+ release induced by noradrenaline, but did not inhibit caffeine-induced Ca2+ release. The peak rise in intracellular Ca2+ following K+, or noradrenaline in physiological saline was unaffected by heparin. The use of reversible permeabilisation may prove a useful approach, allowing introduction of a variety of membrane-impermeant blockers of second messenger systems into intact resistance arteries.

The action of amlodipine on human subcutaneous resistance arteries studied in vitro.

The effect of amlodipine, a dihydropyridine calcium antagonist which is largely ionized at physiological pH, was studied in human resistance arteries. Resistance arteries were isolated from subcutaneous fat and isometric force measured in a myograph. Amlodipine inhibited depolarization-induced contractions in a time- and concentration-dependent manner. The potency of amlodipine was markedly increased by depolarization of the resistance arteries by a physiological saline containing high potassium (40 mM) during exposure to amlodipine. The onset and offset of amlodipine-induced inhibition in these arteries was slow, but concentration dependent. Depolarization markedly increased the rate of onset of inhibition. The increase in potency of amlodipine under depolarized conditions could largely be accounted for by the increased rate of association of the drug. Possible use-dependence of amlodipine was also examined in comparison with verapamil. The efficacy of both verapamil and amlodipine was increased in vessels which were repeatedly depolarized compared with vessels which were only activated once. This effect was more marked for verapamil than for amlodipine. The action of amlodipine in human resistance arteries is slow, shows marked voltage-dependence and, to a lesser degree, some use-dependence. These properties may be important in understanding the action of amlodipine in vivo.