1 In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels. 2 Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was ®rst signi®cant at a concentration of 1 nM (115+3% of control, n=11) with 1 mM, the highest concentration tested, increasing the rate to 153+4% of control (n=9). 3 Repetitive 5 min applications of substance P (1 mM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and signi®cantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively. 4 The competitive antagonist of tachykinin receptors, spantide (5 mM) and the speci®c NK 1 receptor antagonist, WIN51708 (10 mM) both prevented the enhancement of constriction rate induced by 1 mM substance P. 5 Endothelial cells loaded with the Ca 2+ sensing¯uophore,¯uo 3/AM did not display a detectable change in [Ca 2+ ] i upon application of 1 mM substance P. 6 Inhibition of nitric oxide synthase by N G nitro-L-arginine (L-NOARG; 100 mM) had no signi®cant e ect on the response induced by 1 mM substance P. 7 The enhancement of constriction rate induced by 1 mM substance P was prevented by the cyclooxygenase inhibitor, indomethacin (3 mM), the thromboxane A 2 synthase inhibitor, imidazole (50 mM), and the thromboxane A 2 receptor antagonist, SQ29548 (0.3 mM). 8 The stable analogue of thromboxane A 2 , U46619 (0.1 mM) signi®cantly increased the constriction rate of lymphangions with or without endothelium, an e ect which was prevented by SQ29548 (0.3 mM). 9 Treatment with pertussis toxin (PTx; 100 ng ml 71 ) completely abolished the response to 1 mM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate. 10 Application of the phospholipase A 2 inhibitor, anti¯ammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 mM), without inhibiting the response to either U46619 (0.1 mM) or acetylcholine (10 mM). 11 The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK 1 receptors coupled by a PTx sensitive G-protein to phospholipase A 2 and resulting in generation of the arachidonic acid metabolite, thromboxane A 2 , this serving as the di usible activator.

1 Experiments were made to investigate mechanisms by which adenosine 5'-trisphosphate (ATP) enhanced vasomotion in mesenteric lymphatic vessels isolated from young guinea-pigs. 2 ATP (10 78 ± 10 73 M) caused a concentration-dependent increase of perfusion-induced vasomotion with the endothelium mediating a fundamental role at low ATP concentrations (10 78 ± 10 76 M).3 The response to 10 76 M ATP showed tachyphylaxis when applied at intervals of 10 min but not at intervals of 20 or 30 min. inhibitors of speci®c G proteins, phospholipase A 2 , cyclo-oxygenase and thromboxane A 2 receptors respectively. 6 ATP simultaneously induced a suramin-sensitive inhibitory response, which was normally masked by the excitatory response. ATP-induced inhibition was mediated by endothelium-derived nitric oxide (EDNO) as the response was abolished by N G -nitro-L-arginine (L-NOARG; 10 74 M), an inhibitor of nitric oxide synthase. 7 We conclude that ATP modulates lymphatic vasomotion by endothelium-dependent and endothelium-independent mechanisms. One of these is a dominant excitation caused through endothelial P 2 purinoceptors which because of an involvement of a pertussis toxin sensitive Gprotein may be of the P 2Y receptor subtype. Their stimulation increases synthesis of phospholipase A 2 and production of thromboxane A 2 , an arachidonic acid metabolite which acts as an endothelium-derived excitatory factor.

The effects of calcitonin generelated peptide (CGRP) on constriction frequency, smooth muscle membrane potential (V m), and endothelial Vm of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1-100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP-1 receptor antagonist CGRP-(8 -37) (1 M) and pertussis toxin (100 ng/ml). This action of CGRP was also blocked by the nitric oxide (NO) synthase inhibitor N G -nitro-Larginine (L-NNA; 10 M), an action that was reversed by the addition of L-arginine (100 M). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA), and ATP-sensitive K ϩ (K ATP ϩ ) channels were all implicated in the endothelium-dependent CGRP response because it was abolished by methylene blue (20-5-isoquinolinesulfonamide-dichloride (H89; 1 M) and glibenclamide (10 M). CGRP (100 nM), unlike acetylcholine, did not alter endothelial intracellular Ca 2ϩ concentration or V m. CGRP (100 nM) hyperpolarized the smooth muscle Vm, an effect inhibited by L-NNA, H89, or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP-(8 -37). CGRP (500 nM) also caused smooth muscle hyperpolarization, an action that was now not blocked by L-NNA (100 M). It was most likely mediated by the activation of the cAMP/PKA pathway and the opening of K ATP ϩ channels because it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e., 1-100 nM), decreases lymphatic constriction frequency primarily by the stimulation of CGRP-1 receptors coupled to pertussis toxin-sensitive G proteins and the release of NO from the endothelium or enhancement of the actions of endogenous NO. At high concentrations (i.e., 500 nM), CGRP also directly activates the smooth muscle independent of NO. Both mechanisms of activation ultimately cause the PKA-mediated opening of K ATP ϩ channels and resultant hyperpolarization. smooth muscle; endothelium; nitric oxide; adenosine 3Ј,5Ј-cyclic monophosphate-dependent protein kinase; vasomotion CALCITONIN GENE-RELATED PEPTIDE (CGRP), like substance P, is a dominant neurotransmitter in vascular sensory nerves and is a potent vasodilator, which may play a role in modulation of total peripheral resistance of the systemic circulation (6,22,30). The pathways by which CGRP produces vasodilation vary between vascular beds. In the majority of blood vessels, application of CGRP has been reported to cause dilation through an endothelium-independent process. These vessels include cat cerebral (16, 48), rat mesenteric (1, 20, 25, 35), rat stomach mucosal (27), rabbit jejunal (34), dog basilar (40), dog lingual (33), and human uterine arteries (5). A primary signal transduction pathway involved in this form of CGRP activation is adenylate cyclase, which increases cAMP and cAMP-dependent protein kinase (PKA)-activating K ϩ channels including ATP-sensitive K ϩ (K ATP ϩ ) channels (37,...

1 The e ects of continuous but asynchronous nerve activity induced by ciguatoxin (CTX-1) on the membrane potential and contraction of smooth muscle cells have been investigated in rat proximal tail arteries isolated in vitro. These e ects have been compared with those produced by the continuous application of phenylephrine (PE). 2 CTX-1 (0.4 nM) and PE (10 mM) produced a maintained depolarization of the arterial smooth muscle that was almost completely blocked by a-adrenoceptor blockade. In both cases, the depolarization was more sensitive to the selective a 2 -adrenoceptor antagonist, idazoxan (0.1 mM), than to the selective a 1 -adrenoceptor antagonist, prazosin (0.01 mM). 3 In contrast, the maintained contraction of the tail artery induced by CTX-1 (0.2 nM) and PE (2 and 10 mM) was more sensitive to prazosin (0.01) mM, than to idazoxan (0.01 mM). In combination, these antagonists almost completely inhibited contraction to both agents. 4 Application of the calcium channel antagonist, nifedipine (1 mM), had no e ect on the depolarization induced by either CTX-1 or PE but maximally reduced the force of the maintained contraction to both agents by about 50%. 5 We conclude that the constriction of the tail artery induced by CTX-1, which mimics the natural discharge of postganglionic perivascular axons, is due almost entirely to a-adrenoceptor activation. The results indicate that neuronally released noradrenaline activates more than one a-adrenoceptor subtype. The depolarization is dependent primarily on a 2 -adrenoceptor activation whereas the contraction is dependent primarily on a 1 -adrenoceptor activation. The links between a-adrenoceptor activation and the voltage-dependent and voltage-independent mechanisms that deliver Ca 2+ to the contractile apparatus appear to be complex.