Potentiation by aminoethylisothiourea of the extra-cellular Ca(2+) component of norepinephrine-induced contraction in rat femoral arteries.

Aminoethylisothiourea (AET) is a potent inhibitor of inducible nitric oxide synthase (NOS). The present study was performed to investigate whether AET and its rearrangement products might modulate vascular contraction independently of its effects as a NOS inhibitor in rat small femoral arteries. AET caused an endothelium-independent increase in contraction induced by norepinephrine (NE). This effect was not affected by either N(omega)-nitro-L-arginine methyl ester, nitro-L-arginine, indomethacin or propanolol, but it was suppressed in Ca(2+)-free medium. AET enhanced extracellular Ca(2+) component of NE-induced contraction, and this effect was prevented by the receptor-mediated Ca(2+) entry blocker, 1-{beta-[3-(p-methoxyphenyl)-propyloxyl]-p-methoxyphenetyl}- 1H-imidaz ole hydrochloride (SK&F 96365), but not by the voltage-dependent Ca(2+) channel blocker, nitrendipine. AET did not alter the response to CaCl(2) in vessels exposed to KCl depolarization. The protein kinase C (PKC) inhibitor, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) (GF 109203X), prevented the potentiating effect of AET on the NE response. AET failed to produce an increase in tone in the presence of NE and GTP in permeabilized arteries. Among the AET rearrangement products, mercaptoethylguanidine produced an endothelium-independent increase in the NE response. 2-aminothiazoline had no effect, and guanidinoethyldisulphide produced relaxation. The effect of mercaptoethylguanidine was dependent on extracellular Ca(+) and was prevented by GF 109203X. These results indicate that AET is able to potentiate the contraction to NE in rat femoral resistance arteries independently of its inhibitory effect on either NOS or cyclo-oxygenase. Its effect occurs via an enhancement of SK&F 96365-sensitive Ca(2+) entry. A PKC inhibitor-sensitive mechanism also appears to be involved in the AET effect. Mercaptoethylguanidine potentiates NE response through a mechanism similar to AET.

Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca(2+) handling of human small arteries.

The mechanisms of Ca(2+) handling and sensitization were investigated in human small omental arteries exposed to norepinephrine (NE) and to the thromboxane A(2) analog U-46619. Contractions elicited by NE and U-46619 were associated with an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), an increase in Ca(2+)-independent signaling pathways, or an enhancement of the sensitivity of the myofilaments to Ca(2+). The two latter pathways were abolished by protein kinase C (PKC), tyrosine kinase (TK), and Rho-associated protein kinase (ROK) inhibitors. In Ca(2+)-free medium, both NE and U-46619 elicited an increase in tension that was greatly reduced by PKC inhibitors and abolished by caffeine or ryanodine. After depletion of Ca(2+) stores with NE and U-46619 in Ca(2+)-free medium, addition of CaCl(2) in the continuous presence of the agonists produced increases in [Ca(2+)](i) and contractions that were inhibited by nitrendipine and TK inhibitors but not affected by PKC inhibitors. NE and U-46619 induced tyrosine phosphorylation of a 42- or a 58-kDa protein, respectively. These results indicate that the mechanisms leading to contraction elicited by NE and U-46619 in human small omental arteries are composed of Ca(2+) release from ryanodine-sensitive stores, Ca(2+) influx through nitrendipine-sensitive channels, and Ca(2+) sensitization and/or Ca(2+)-independent pathways. They also show that the TK pathway is involved in the tonic contraction associated with Ca(2+) entry, whereas TK, PKC, and ROK mechanisms regulate Ca(2+)-independent signaling pathways or Ca(2+) sensitization.

Mechanism of Ca2+ release and entry during contraction elicited by norepinephrine in rat resistance arteries.

The intracellular Ca2+ stores and the mechanisms of Ca2+ entry produced by norepinephrine (NE) were investigated in small mesenteric resistance arteries of the rat. In Ca2+-free medium, NE (10 microM) elicited a transient increase in both intracellular free Ca2+ concentration ([Ca2+]i) and tension that were both drastically reduced by caffeine and only partially reduced by the two sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, despite the presence of SERCA2a and SERCA2b isoforms in the medial smooth muscle layer of the artery. After depletion of intracellular Ca2+ stores with 10 microM NE, addition of exogenous CaCl2 (2.5 mM) produced large and sustained increases in both [Ca2+]i and contraction of the arteries provided that the agonist was continuously present. In these conditions, the responses to CaCl2 were inhibited by the voltage-dependent Ca2+ entry blocker nitrendipine (1 microM), the putative inhibitor of receptor-operated Ca2+ entry SKF-96365 (30 microM), and NiCl2 (1 mM). The inhibition produced by SKF-96365 and NiCl2 was greater than that of nitrendipine. Also, the responses to CaCl2 were greatly reduced or abolished in the presence of the Na+/Ca2+ exchanger inhibitors 1,3-dimethyl-2-thiourea, 3',4'-dichlorobenzamil, MgCl2, and amiloride or after omission of NaCl in the medium. Also, protein kinase C inhibitors, calphostin C and staurosporine, and tyrosine kinase inhibitors, genistein and tyrphostin 23, both reduced the responses to CaCl2. The inhibitory effect of protein kinase C inhibitor and tyrosine kinase were additive. These results suggest that NE releases Ca2+ from intracellular stores that are caffeine sensitive and partially sensitive to SERCA inhibitors. They indicate that in addition to Ca2+ influx via nitrendipine-sensitive and SKF-96365-sensitive channels, Na+/Ca2+ exchanger participates in the CaCl2-induced contraction produced in NE-exposed vessels. The pathway leading to Ca2+ entry probably involves tyrosine kinase and protein kinase C. All the above mechanisms require ongoing receptor stimulation.

Role of endothelial nitric oxide in the response to angiotensin II of small mesenteric arteries of the rat.

The role of endothelium-derived nitric oxide (NO) in the vascular contractile response to angiotensin II (Ang II) has been investigated in isolated small mesenteric resistance arteries of the rat. Both contraction and intracellular Ca2+ ion concentration ([Ca2+]i) were monitored in vessels, with and without functional endothelium, which were exposed to physiological salt solution containing 25 mM KCl. Ang II induced concentration-dependent contractile responses and increases in [Ca2+]i which, at the concentration giving the maximal response (10 nM), were not sustained in arteries with functional endothelium; however, the presence of a functional endothelium did not modify the peak responses. Ang II did not increase the cyclic guanosine 3',5'-monophosphate content of the tissue nor did it induce relaxation in arteries precontracted with 3 microM noradrenaline. The decline of the Ang II responses was suppressed by removal of the endothelium or by exposure of arteries with endothelium to either the NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (300 microM), or the cyclic GMP-dependent protein kinase inhibitor, Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (30 microM). On the other hand, the NO donor SIN-1 (3-morpholino-sydnonimine, 10 microM) accelerated the decline in [Ca2+]i and contraction. These results show that endothelium-derived NO does not affect the magnitude of the phasic element of the response to Ang II, but is involved in the rapid attenuation of the tonic component. Activation of cyclic GMP-dependent protein kinase accounts for this effect of endothelium-derived NO.