Receptors linked to polyphosphoinositide hydrolysis stimulate Ca2+ extrusion by a phospholipase C-independent mechanism.

In A7r5 cells with empty intracellular Ca(2+) stores in which the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) had been increased by capacitative Ca(2+) entry, stimulation of receptors linked to phospholipase C (PLC), including those for Arg(8)-vasopressin (AVP) and platelet-derived growth factor (PDGF), caused a decrease in [Ca(2+)](i.) This effect was further examined in a stable variant of the A7r5 cell line in which the usual ability of hormones to stimulate non-capacitative Ca(2+) entry is not expresssed. In thapsigargin-treated cells, neither AVP nor PDGF affected capacitative Mn(2+) or Ba(2+) entry, but both stimulated the rate of Ca(2+) extrusion, and their abilities to decrease [Ca(2+)](i) were only partially inhibited by removal of extracellular Na(+). These results suggest that receptors linked to PLC also stimulate plasma membrane Ca(2+) pumps. Activation of protein kinase C by phorbol 12, 13-dibutyrate (PDBu, 1 microM) also caused a decrease in [Ca(2+)](i) by accelerating Ca(2+) removal from the cytosol; the effect was again only partially inhibited by removal of extracellular Na(+). An inhibitor of PKC, Ro31-8220 (10 microM), abolished the ability of PDBu to decrease [Ca(2+)](i), without affecting the response to maximal or submaximal concentrations of AVP. Similar experiments with PDGF were impracticable because Ro31-8220, presumably by inhibiting the tyrosine kinase activity of the PDGF receptor, abolished all responses to PDGF. U73122 (10 microM), an inhibitor of PLC, completely inhibited PDGF- or AVP-evoked Ca(2+) mobilization, without preventing either stimulus from causing a decrease in [Ca(2+)](i). We conclude that receptors coupled to PLC, whether via G-proteins or protein tyrosine kinase activity, also share an ability to stimulate the plasma membrane Ca(2+) pump via a mechanism that does not require PLC activity.

A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin.

1. Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. 2. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP3) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca2+ mobilization but not Sr2+ entry. 3. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP-evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. 4. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 microM Gd3+, but not by 1 microM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. 5. AVP-evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. 6. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non-capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. 7. We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase.

Mechanisms of acute vasodilator response to bacterial lipopolysaccharide in the rat coronary microcirculation.

1. In this study the mechanisms of the acute vasodilator action of bacterial lipopolysaccharide (LPS) were investigated in the rat Langendorff perfused heart. 2. Infusion of LPS (5 microg ml(-1)) caused a rapid and sustained fall in coronary perfusion pressure (PP) of 59 +/- 4 mmHg (n = 12) and a biphasic increase in NO levels determined in the coronary effluent by chemiluminescent detection. Both the fall in PP and the increase in NO release were completely abolished (n = 3) by pretreatment of hearts with the NO synthase inhibitor L-NAME (50 microM). 3. LPS-induced vasodilatation was markedly attenuated to 5 +/- 4 mmHg (n 3) by pretreatment of hearts with the B2 kinin receptor antagonist Hoe-140 (100 nM). 4. Vasodilator responses to LPS were also blocked by brief pretreatment with mepacrine (0.5 microM, n = 3) or nordihydroguaiaretic acid (0.1 microM, n = 4) and markedly attenuated by WEB 2086 (3 microM, n = 4). 5. Thirty minutes pretreatment of hearts with dexamethasone (1 nM), but not progesterone (1 microM), significantly modified responses to LPS. The action of dexamethasone was time-dependent, having no effect when applied either simultaneously with or pre-perfused for 5 min before the administration of LPS but inhibiting the response to LPS by 91 +/- 1% (n = 4) when pre-perfused for 15 min. The inhibition caused by dexamethasone was blocked by 15 min pretreatment with the glucocorticoid receptor antagonist RU-486 (100 nM) or by 2 min pre-perfusion of a 1:200 dilution of LCPS1, a selective antilipocortin 1 (LC1) neutralizing antibody. 6. Treatment with the protein synthesis inhibitor, cycloheximide (10 microM, for 15 min) selectively blunted LPS-induced vasodilatation, reducing the latter to 3 +/- 5 mmHg (n = 3), while having no effect on vasodilator responses to either bradykinin or sodium nitroprusside. 7. These results indicate that LPS-induced vasodilatation in the rat heart is dependent on activation of kinin B2 receptors and synthesis of NO. In addition, phospholipase A2 (PLA2) is activated by LPS resulting in the release of platelet-activating factor (PAF) and lipoxygenase but not cyclo-oxygenase products. These effects are dependent on de novo synthesis of an intermediate protein which remains to be identified.

Endothelin ETA and ETB receptors mediate vascular smooth-muscle contraction.

Endothelin (ET) ETA receptors on vascular smooth muscle are believed to mediate the vasoconstrictor effects of ET isopeptides, and ETB receptors on the endothelium are thought to mediate the vasodilator effects. This study has investigated the receptors mediating endothelin-induced contraction of isolated ring preparations of rat thoracic aorta (RTA) and rabbit carotid artery (RCA), pulmonary artery (RPA), and jugular vein (RJV). In RTA and RCA, ET-1 (EC50 4.5 and 5.2 nM, respectively) was 82- and 108-fold, respectively, more potent than ET-3, whereas the ETB receptor-selective agonists sarafotoxin S6c (S6c) and Ala1,3,11,15-ET-1 (4-Ala-ET-1) were without effect up to > or = 1 microM. In contrast, in RPA and RJV, ET-1 (EC50 3.1 and 0.7 nM, respectively) and ET-3 (EC50 4.4 and 0.9 nM, respectively) were equipotent, and 4-Ala-ET-1 (EC50 10.7 and 2.1, respectively) and S6c (EC50 0.4 and 0.1 nM, respectively) were potent contractile agonists. The ETA receptor antagonist BQ123 (D-Val-Leu-D-Trp-D-Asp-Pro) competitively antagonized the effects of ET-1 in RTA and RCA (pA2 values 6.9 +/- 0.1 and 6.8 +/- 0.2, respectively) but did not antagonize (at 10 microM) contractions to ET-1, ET-3, or 4-Ala-ET-1 in RPA and RJV. In conclusion, contraction of vascular smooth muscle by endothelins can be mediated by both ETA and ETB receptors.

Endothelin ETA and ETB receptors mediate vascular smooth muscle contraction.

1. We have investigated the receptors mediating endothelin-induced contraction of rabbit isolated jugular vein (RJV) and rat isolated thoracic aorta (RTA). 2. Endothelin-1 (ET-1) and endothelin-3 (ET-3) contracted RJV preparations with similar potency (EC50 values approximately 1 nM), whereas, ET-1 (EC50:4.5 nM) was approximately 80 fold more potent than ET-3 in contracting RTA. In addition, the ETB receptor-selective agonist [Ala1,3,11,15]ET-1 contracted RJV (EC50:2.1 nM) but not RTA. 3. The ETA receptor antagonist, BQ123, competitively antagonized (pA2 6.93) the contraction of RTA produced by ET-1, but had no effect (at 10 microM) on the contractile effects of either ET-1, ET-3 or [Ala1,3,11,15]ET-1 in RJV. 4. These data suggest that both ETA and ETB receptors can mediate vascular smooth muscle contraction.