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Parathyroid hormone-related protein (PTHrP) was originally found as a tumor-derived vasoactive factor and has also been known to produce significant relaxation of vascular smooth muscles. Thus effects of PTHrP-(1-34), a PTH receptor-binding domain, on spontaneous lymphatic pump activity was investigated in isolated pressurized lymph vessels of mice. Low concentrations (1 × 10−10 and 3 × 10−10 M) of PTHrP-(1-34) dilated lymph vessels and reduced the frequency of pump activity, whereas high concentrations (1 × 10−9 to 1 × 10−8 M) of PTHrP-(1-34) caused dilation with cessation of the lymphatic pump activity. N ω-nitro-l-arginine methyl ester (l-NAME; 3 × 10−5 M) but not indomethacin (1 × 10−5 M) significantly reduced the PTHrP-(1-34)-induced inhibitory responses of the lymphatic pump activity. In the presence of l-NAME (3 × 10−5 M) and l-arginine (1 × 10−3 M), the l-NAME-induced inhibition in the PTHrP-(1-34)-mediated responses was significantly reduced. Glibenclamide (1 × 10−6 M) significantly suppressed the inhibitory responses of the lymphatic pump activity induced by PTHrP-(1-34) and S-nitroso- N-acetyl-penicillamine. The PTHrP-(1-34)-mediated inhibitory responses were significantly reduced by treatment with PTHrP-(7-34) (1 × 10−7 M). These results suggest that PTHrP-(1-34) inhibits spontaneous pump activity of the isolated lymph vessels via PTH receptors and that production and release of endogenous nitric oxide and activation of ATP-sensitive K+ channels in the lymph vessels contribute to the PTHrP-(1-34)-mediated inhibitory responses of the lymphatic pump activity.

A mode of action of endothelin (ET) on spontaneous contractions was investigated in ring preparations of isolated bovine mesenteric lymphatics. ET-1 at concentrations between 10−10and 10−9M caused a dose-dependent increase in the frequency of spontaneous contractions. The specific ETA-receptor antagonist BQ-123 (5 × 10−7M) caused a significant inhibition of the ET-1-induced positive chronotropic effect in the ring preparations with and without the endothelium. Mechanical denudation of the lymphatic endothelial cells produced a significant potentiation of the ET-induced positive chronotropic effect. BQ-3020 (10−8–10−7M), a selective ETB-receptor agonist, induced dose dependently negative chronotropic and inotropic effects on the spontaneous contractions in the ring preparations with intact endothelium. Mechanical removal of the endothelium caused a significant reduction of the BQ-3020-induced negative chronotropic and inotropic effects. The ET-1-induced positive chronotropic effect was potentiated by pretreatment with Nω-nitro-l-arginine methyl ester (l-NAME) (10−5M) but unaffected by aspirin (10−5M). Additional treatment with l-arginine (10−4M) completely reversed the l-NAME-mediated potentiation of the ET-induced chronotropic effect. These results suggest that stimulation of ETAreceptors on the lymphatic smooth muscles causes a positive chronotropic effect on the spontaneous contractions, and stimulation of ETBreceptors on the lymphatic endothelial cells induces a release of nitric oxide, which results in the chronotropic and inotropic effects on spontaneous contractions in isolated bovine mesenteric lymphatics.

To study flow-mediated responses in a conduit vein, we investigated the physiological characteristics of endothelium-dependent acetylcholine (ACh)- and flow-induced relaxations using a conventional bioassay cascade. Cylindrical segments isolated from canine common carotid arteries and external jugular veins were perfused at a constant mean flow rate ranging from 1 to 8 ml/min. Endothelium-derived nitric oxide (NO) activity in perfusion effluent through the arterial and venous segments was measured by relaxation of endothelium-denuded arterial rings and arterial and/or venous rings precontracted by prostaglandin F2 alpha, respectively. Stimulation by a flow rate of 8 ml/min on the arterial and venous endothelial cells produced approximately 60 and 20% of the maximum relaxation in the arterial and venous rings, respectively. ACh (10(-6) and 10(-5) M) perfused through the arterial and venous segments with endothelium caused dose-related relaxations of both bioassay rings. The ACh- and flow-induced relaxations were completely reduced by mechanical removal of the endothelial cells. Pretreatment with 5 x 10(-5) M NG-nitro-L-arginine methyl ester (L-NAME) produced a significant reduction of the ACh- and flow-induced vasodilation. Additional treatment with 10(-4) M L-arginine significantly reversed the L-NAME-induced inhibition of ACh-induced relaxation but had no effect on flow-induced relaxation. When the flow rate was increased from 2 to 4 ml/min, the same concentrations of ACh produced larger dose-related relaxations than those obtained at a flow rate of 2 ml/min. Pretreatment with 25 U/ml superoxide dismutase caused no significant effect on the flow-mediated potentiation of ACh-induced relaxation. These findings suggest that venous endothelial cells of canine large vein are able to produce and release NO by stimulation of increased flow or ACh to a significantly lesser extent compared with the artery and that ACh-induced vasodilation is potentiated by an increase in shear stress up to approximately 4 dyn/cm2 loaded on the endothelial cells.

5-Hydroxytryptamine (5-HT, 0.01-100 microM) and 5-carboxamidotryptamine (5-CT, 0.001-10 microM) produced dose-related relaxations in strips cut from monkey popliteal lymph nodes precontracted with a perfusion of Krebs bicarbonate solution containing 80 mM KCl. These 5-HT agonists caused no significant effect on the basal tone of the lymph node strips. The 5-HT-induced relaxation is competitively antagonized by pretreatment with a selective 5-HT1-like receptor antagonist, methiothepin (0.01-0.1 microM). Schild plot analysis showed that the pA2 value and slope of methiothepin against 5-HT were 8.80 +/- 0.11 and 0.99 +/- 0.07 (n = 6), respectively. Pretreatment with methysergide (0.01-0.1 microM) significantly attenuated the 5-HT-induced relaxation of the strips. On the other hand, treatment with ketanserin (0.01-0.1 microM) and ICS-205-930 (0.01-0.1 microM) caused no significant effect on the 5-HT-or the 5-CT-induced relaxation. The 5-HT-induced relaxation was significantly reduced by 10 microM NG-monomethyl-L-arginine, which was reversed by 1 mM L-arginine. The relaxation in the lymph node strips was also significantly reduced by treatment with 10 microM methylene blue but not with 30 microM aspirin. These results suggest that 5-HT1-like receptors exist in the monkey popliteal lymph nodes. Stimulation of these receptors produces an endogenous nitric oxide (NO)-dependent relaxation in lymph node smooth muscle through an activation of cytosolic guanylate cyclase in the cells.

The effects of acetylcholine (ACh) on spontaneous contractions in isolated bovine mesenteric lymph vessels were investigated. ACh ranging from 3 x 10(-8) M to 10(-5) M produced dose-dependent negative chronotropic and inotropic effects on the spontaneous contractions. In the lymph vessels without endothelium, ACh at the same concentration range had no significant effect on the spontaneous contractions. Atropine (10(-9) and 10(-8) M) caused a parallel shift to the right of the dose-chronotropic response curve for ACh. The pA2 value of atropine to ACh in the negative chronotropic effect was 8.90 +/- 0.20 (n = 6). Aspirin (10(-5) M) produced no significant inhibition of the ACh-induced negative chronotropic and inotropic effects. NG-monomethyl-L-arginine (L-NMMA; 3 x 10(-5) M) significantly suppressed the ACh-induced negative responses on spontaneous contractions. In the same lymphatic segments, L-arginine (10(-4) M) reversed completely the inhibition by L-NMMA of the ACh-induced responses. These results suggest that low concentrations of ACh produce negative chronotropic and inotropic effects on spontaneous contractions in bovine mesenteric lymphatics and that the responses may be mediated by nitric oxide or its related compound released from the endothelial cells through activation of low-affinity muscarinic receptors.

Effects of prostaglandin F2 alpha (PGF2 alpha) on isolated monkey and dog cerebral arteries were investigated to reevaluate PGF2 alpha's possible action on the endothelium. Low concentrations of PGF2 alpha ranging from 10(-11) to 10(-8) M produced a dose-dependent relaxation in the monkey arteries. PGF2 alpha (10(-7) M) produced a transient contraction followed by a small relaxation, whereas higher concentrations (greater than 10(-6) M) of PGF2 alpha induced only contractions. The PGF2 alpha-induced relaxation was not observed in the canine cerebral arteries. The PGF2 alpha-induced relaxation of the monkey cerebral arteries was not affected by treatment with 10(-7) M propranolol, 10(-7) M atropine, or 10(-6) M cimetidine. In monkey cerebral arteries without endothelium, PGF2 alpha in concentrations ranging from 10(-11) to 10(-6) M caused no relaxation. Treatment with 5 X 10(-5) M aspirin, 3 X 10(-5) M NG-monomethyl-L-arginine, and 10(-5) M oxyhemoglobin significantly suppressed the PGF2 alpha-induced relaxation. These results suggest that low concentrations of PGF2 alpha may produce an endothelium-dependent relaxation in monkey cerebral arteries and that the relaxation may be mediated by release of both endogenous vasodilative prostaglandins and endothelium-derived relaxing factor from endothelial cells.

Ring segments of dog thoracic ducts precontracted with a high concentration of norepinephrine (NE) relaxed in a concentration-dependent manner in response to acetylcholine (ACh) or sodium nitroprusside (SNP). Pretreatment with atropine inhibited the ACh-induced relaxation in a competitive manner. The Schild plot showed a slope of 1.1 +/- 0.2 and a pA2 value of 10.4 +/- 0.4 (n = 6 ring segments). Removal of endothelium caused a complete inhibition of the ACh-induced relaxations in precontracted dog thoracic ducts. ACh, which failed to relax precontraction of the ring segment when mounted separately, induced relaxation in the same preparation when it was mounted as a "sandwich" with the longitudinal strip. The ACh-induced relaxations in the lymphatic preparations with endothelium were suppressed or abolished by pretreatment with oxyhemoglobin (10(-6) and 10(-5) M), methylene blue (10(-6) and 10(-5) M), and NG-monomethyl-L-arginine (3 x 10(-5) M), but the relaxations were unaffected by aspirin (10(-5) M). Pretreatment with methylene blue (10(-5) M) also caused a significant reduction of the SNP-induced relaxations in the precontracted thoracic ducts. It may be concluded that ACh-induced relaxation in dog thoracic ducts precontracted with NE is mediated by high-affinity muscarinic receptors in the lymphatic endothelial cells. Also, stimulation of the endothelial muscarinic receptors liberates a transferable endothelium-derived relaxing factor, which results in the relaxation of the lymphatic smooth muscles via the accumulation of cellular guanosine 3',5'-cyclic monophosphate.

The mode of action of atrial natriuretic peptide (ANP) on bovine mesenteric lymphatics was investigated by recording isometric tensions in isolated cylindrical segments. ANP in concentrations from 5 to 30 ng/ml caused dose-related decreases in the rhythm and amplitude of spontaneous contractions. No tachyphylaxis was observed in the ANP-induced responses in lymph vessels. Addition of ANP in a low concentration ranging from 3 to 100 ng/ml produced a dose-dependent relaxation in the lymphatic preparations precontracted by 10(-7) M bradykinin. The ANP-induced relaxation was not modified by pretreatment with 5 x 10(-7) M propranolol, 5 x 10(-7) M atropine, 10(-6) M cimetidine, 5 x 10(-5) M aspirin, or 10(-5) M ouabain. The mechanical rubbing of endothelial cells in the lymph vessels caused no significant effect on the ANP-induced relaxation. The relaxation, however, was significantly reduced by pretreatment with 10(-5) M methylene blue. These results suggest that ANP in a low concentration seems to inhibit lymph transport through a reduction of spontaneous contractions and a marked relaxation of lymphatic smooth muscles in bovine mesenteric lymphatics and that ANP may produce the relaxation through synthesis of guanosine 3',5' cyclic monophosphate, independent of the lymphatic endothelium.

Pressure-volume and pressure-radius relationships in lymphangions isolated from bovine mesenteric lymphatics were similar in pattern with those in the vein. Circumferential modulus of elasticity of the lymphatics ranged from 4.2 x 10(4) tatic walls. The contractile force increased in early stages of distension and decreased after an optimal intraluminal pressure was attained. The spontaneous activity was also affected by the rate of wall deformation. The pacemaker site of spontaneous activity seemed to be in the wall in the immediate vicinity of the inlet valve of a lymphangion. The activity propagated with a velocity of 4-5 mm/s. Ejection fraction of a lymphangion was between 45 and 65%. The endurance limit of the lymphatic valve was 68.4 +/- 7.6 cmH2O in specimens of about 3 mm in outer diameter. These findings suggested that lymphatic smooth muscle seemed to play a major role in elastic behavior of the wall and in regulation of the spontaneous activity, thereby affecting significantly passive and active lymph transport.