Alex L. Loeb

Mechanisms of endothelium-dependent vascular smooth muscle relaxation

In recent years, it has become apparent that several agents which relax blood vessels do so by endothelium-dependent mechanisms. Among the first group of agents shown to modulate vascular tone via an interaction with the endothelium were angiotensin II and bradykinin. Subsequent studies indicated that the endothelium responded to these polypeptides by producing and releasing prostacyclin (PGI,) which, in turn, could modulate the change in vascular tone. In 1980, Furchgott and Zawadzki [l] reported that the endothelium was requisite for acetylcholine (Ach)-induced vasodilation. The relaxing factor, however, was not PG12 since relaxation was not blocked by cyclooxygenase inhibitors (i.e. indomethacin or aspirin; Fig. 1). Since this initial report, numerous laboratories have confirmed the endothelium-dependence of Ach and a variety of other vasodilators (for review, see Refs. 2-4). Dr. Furchgott and his colleagues coined the term “endothelium-delved relaxing factor” (EDRF) for the substance(s) which mediates the decrease in smooth muscle contraction. Numerous techniques have been used to remove the endothelium from the intima of arteries: (1) mechanical (i.e. rubbing, blotting with filter paper, balloon catheter, frayed nylon cord, adhesion to glass coverslip), (2) physical (blowing air on the intima, hypotonic perfusion, perfusion for up to 10min with distilled H20, exposure to hype~onic medium with added IV), (3) chemical (removal of Ca2+ and addition of chelators-EGTA/EDTA), and (4) enzymatic (perfusion or incubation with trypsin or collagenase). In all cases, coincident with endothelium disruption as verified by microscopy, is the loss of a relaxation response observed in conduit and muscular arteries to the agents summarized in Table 1. In arteries, the endothelium becomes increasingiy difficult to remove as the vessel diameter decreases. Some laboratories using the techniques above have been unsuccessful in removing the endothelium from isolated mesenteric arterioles (75-100 pm in diameter). In the perfused mesenteric arcade of the rat, exposure to distilled water for 5-10 min was required to destroy the endothelium. Obviously, when harsh measures are required to remove the endothelium, the medial layer of smooth muscle may also be

Role of calcium in endothelium-dependent relaxation of arterial smooth muscle

Endothelium-dependent relaxation was studied in rings of rabbit thoracic aorta. Relaxation responses were induced with methacholine, the calcium ionophore A23187 and maitotoxin before and after removal of Ca++ from the external medium; in the presence of calcium-channel entry blockers (verapamil and nifedipine); or with trifluoperazine. Deletion of Ca++ greatly impaired responses to all 3 agonists while trifluoperazine only blocked cholinergic-induced relaxation. The calcium-channel blockers had effects that were concentration- and time-dependent, but their action included blockade of A23187. Cytosolic-free Ca++ concentrations were measured in cultured endothelial cells after incubation of the cells with 10 microM Fura-2/AM or 50 microM Quin 2/AM. Bradykinin (1 X 10(-10) to 1 X 10(-7) M) and melittin (0.5 to 5 micrograms/ml) caused dose-dependent increases in intracellular Ca++ with maximal responses at 3 X 10(-8) M and 3 micrograms/ml, respectively. Both agents were able to induce an increase in cytosolic-free Ca++ in the presence of EGTA (1.5 X 10(-3) M) or verapamil (1 X 10(-5) M). The plateau phase of the Ca++ transient appeared to be modified slightly by verapamil, while the peak responses and plateau were attenuated by 0 Ca++/EGTA. To assess a function of the endothelium, production of endothelium-derived relaxing factor (EDRF) was studied in cells grown on microcarrier beads superfused in a column, and the column effluent was bioassayed on aortic rings.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-derived relaxing factor release associated with increased endothelial cell inositol trisphosphate and intracellular calcium

The release of eicosanoids and endothelium-derived relaxing factor (EDRF) from endothelial cells is thought to involve a calcium-dependent step. Using cultured bovine aortic endothelial cells as a model system, we have examined the relation between agonist-induced changes in inositol polyphosphates and calcium levels within the endothelial cells and extracellular calcium on EDRF release. In a superfusion-cascade system, EDRF was detected by the relaxation of a rabbit aortic ring without endothelium suspended beneath a column of cultured endothelial cells. Endothelial cell stimulation by bradykinin or melittin induced dose-dependent relaxation of the bioassay ring. In addition, bradykinin and melittin stimulated an increase in intracellular calcium concentration in fura-2 loaded endothelial cells and an increase in inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in cells prelabeled with 3H-myoinositol. Bradykinin stimulation produced transient increases in Ins(1,4,5)P3, fura-2 fluorescence and transient EDRF release. Melittin stimulation induced more prolonged release of EDRF from the endothelial cell column, which was correlated with sustained increases in the fura-2 signal and the level of Ins(1,4,5)P3. Omission of calcium from the cell superfusate attenuated, but did not eliminate, bradykinin-induced EDRF release and the calcium transient, whereas the melittin-induced responses were only slightly attenuated. Endothelial cells clearly demonstrate receptor-activation of phospholipase C and release of sequestered calcium from subcellular sites in response to Ins(1,4,5)P3. These results imply that EDRF release is correlated with increased intracellular calcium levels seen in the absence of extracellular calcium. However, sustained release of EDRF does require influx of extracellular calcium via an undefined mechanism.

Changes in vascular endothelium and its function in systemic arterial hypertension

The various functions of arterial endothelium may be altered during pulmonary and arterial hypertension. Changes in the endothelium (or function) associated with hypertension are described. In both acute and chronic hypertension, permeability of the endothelium is enhanced. During the acute phase of hypertension, hyperplasia (cell replication) of the endothelium occurs while cell hypertrophy (enlarged cell size) and an increase in homocellular tight junctions are associated with sustained elevations of blood pressure. Endothelium may contribute to the increase in smooth muscle mass or cell number reported with various models of hypertension. Increased endothelial uptake or metabolism of norepinephrine and serotonin occurs during hypertension. The biotransformation of adenine nucleotides and various peptides by the endothelium is not altered by hypertension. Synthesis of prostacyclin is enhanced in the spontaneously hypertensive and Goldblatt hypertensive rat. Metabolism of prostaglandin E2, prostaglandin F2 alpha and prostacyclin by prostaglandin 15-hydroxydehydrogenase is impaired in the genetic models. Responses to endothelium-dependent vasodilators are impaired in acute and chronic models of hypertension. Production of relaxing factor by the endothelium is not inhibited, but rather the vascular smooth muscle fails to respond. Acute, severe hypertension potentiates the response to serotonin, presumably by attenuating the release or response to relaxing factor(s). In the aorta of the spontaneously hypertensive rat, the endothelium releases a constricting factor in response to acetylcholine. Pulmonary arterial endothelium (and other vessels) releases a vasoconstrictor that is blocked by inhibitors of cyclooxygenase. It is not clear whether this pressor factor is thromboxane A2. Cultured endothelial cells release a polypeptide that contracts arteries; however, any relation to hypertension is not known.

Potentiation of platelet aggregation by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (1 pM-100 nM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 microM)- and ADP (2 or 10 microM)-induced aggregation. Maximum stimulation occurred between 10 to 100 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the shape change. Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, affected platelet activation and cyclic nucleotide levels. Sodium nitroprusside (1 microM) inhibited ADP, but not thrombin or epinephrine-induced aggregation. Both sodium nitroprusside (1 microM) and ANP (10 nM) increased cyclic GMP levels by 80% and 37%, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77% increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function.

Antihypertensive drugs inhibit hypertension-associated aortic DNA synthesis in the rat.

The effect of antihypertensive drug treatment on aortic DNA synthesis was examined in rats with two-kidney, one clip renal hypertension and in spontaneously hypertensive rats (SHR). In two-kidney, one clip hypertensive rats, hypertension developed over a 2-week period. Four days after clipping the renal artery, during the onset of hypertension, there was an increase in aortic DNA synthesis. Aortic DNA synthesis was also increased 3 weeks later, when hypertension had been established. Captopril, hydralazine, and verapamil were each able to prevent the increase in aortic DNA synthesis and the rise in blood pressure when given throughout the first 5 days of the developing phase of hypertension, or when given to rats with established hypertension. Drug treatment of sham-operated rats had no significant effect on DNA synthesis, although blood pressure was decreased. There were no differences in blood pressure or aortic DNA synthesis in 4-week-old SHR, as compared with age-matched Wistar-Kyoto (WKY) controls or normal Wistar rats. At 17 weeks of age, when hypertension was established, aortic DNA synthesis was significantly enhanced in the SHR. Captopril or hydralazine treatment was able to reduce blood pressure and DNA synthesis to levels seen in the WKY. At 21 weeks of age, DNA synthesis in the SHR had declined to the same levels as in the WKY. Captopril, hydralazine, and verapamil may have a common ability to reduce intracellular calcium and therefore inhibit DNA synthesis. In support of this, ouabain treatment, which increases intracellular calcium by inhibiting the Na+-K+ pump, produced a significant increase in the rate of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of Cultured Cells to Study the Relationship Between Arachidonic Acid and Endothelium-Derived Relaxing Factor

We have used mixed- and co-cultures of endothelial and vascular smooth muscle cells to investigate the role of phospholipase activation and arachidonic acid metabolites in the production of endothelium-derived relaxing factor (EDRF). Inhibition of phospholipase A2 with para-bromophenacyl bromide, dexamethasone or quinacrine, alone or in combination, blocked arachidonate release by 50%-60% but had no effect on EDRF production as assessed by cyclic GMP accumulation in mixed- or co-cultures of endothelial and vascular smooth muscle cells. Inhibition of the phospholipase C-diacylglycerol (DAG) lipase pathway of arachidonate release by the DAG lipase inhibitor RHC-80267 also caused partial inhibition of arachidonate release and had no effect on EDRF. When both phospholipase A2 and phospholipase C pathways for arachidonate mobilization were inhibited (dexamethasone + RHC 80267), arachidonate release was totally inhibited while EDRF release remained intact. We conclude that neither phospholipase activation nor arachidonate mobilization is required for EDRF release from cultured bovine endothelial cells.

Endothelium-dependent relaxation is independent of arachidonic acid release

Endothelium-dependent relaxation is mediated by the release from vascular endothelium of an endothelium-derived relaxing factor (EDRF). It is not clear what role arachidonic acid has in this process. Inhibition of phospholipase A2, and diacylglycerol lipase in cultured bovine aortic endothelial cells caused a marked reduction in agonist-induced arachidonic acid release from membrane phospholipid pools, and complete inhibition of prostacyclin production. EDRF release, assayed by measuring endothelium-dependent cGMP changes in mixed endothelial-smooth muscle cell cultures, was not inhibited under these conditions. In fact, EDRF release in response to two agonists, melittin and ATP, was actually increased in cells treated with phospholipase A2 inhibitors. In addition, pretreatment of rats with high-dose dexamethasone, an inhibitor of PLA2, did not attenuate endothelium-dependent relaxation in intact aortic rings removed from the animals, or depressor responses in anesthetized animals induced by endothelium-dependent vasodilators. In summary, inhibition of arachidonic acid release from membrane phospholipid pools does not attenuate endothelium-dependent relaxation in rats, or the release and/or response to EDRF in cultured cells.

Endothelium-derived Relaxing Factor Release from Cultured Endothelial Cells Does Not Require Phospholipase Activation or Arachidonate Mobilization

A large body of literature supports the hypothesis that endothelium-derived relaxing factor (EDRF) is a nonprostanoid metabolite of arachidonic acid. These studies have largely involved correlation of inhibition of lipoxygenase or cytochrome P450 pathways for arachidonate metabolism and inhibition of phospholipase activation with decreased endothelium-dependent relaxation of vascular rings. This hypothesis has been questioned because of the nonspecificity of the inhibitors employed, and their uncertain site of action.v2 We used mixed and cocultures of endothelial and vascular smooth muscle cells to investigate the role of phospholipase activation and arachidonate release in the production of EDRF. Bovine aortic or pulmonary artery endothelial cells and rat vascular smooth muscle cells were prepared as previously described. The ability of the phospholipase A2 inhibitors parabromophenacyl bromide (PBPB; 1 x M), dexamethasone (DEX; 1 x M), and quinacrine (QUIN; 1 x lo- M) as well as the diacylglycerol lipase inhibitor RHC 80267 (4 x M) to inhibit melittin-stimulated arachidonate release from cultured endothelial cells and melittin-stimulated EDRF release from cocultures was determined. Arachidonate release was assessed by measuring the amount of 3H-arachidonate or 6-keto-prostaglandin Fl, released into fresh medium in response to melittin stimulation (2 Ng/ml) in the presence or absence of one or more of the lipase inhibitors and was quantified as percent of control. EDRF release was quantified by determining the amount of cyclic GMP accumulation in coor mixed cultures in response to rnelittin stimulation (2 pg/ml) in the presence or absence of one or more lipase inhibitors (HC1 extraction followed by radioimmunoassay for cyclic GMP). As shown in TABLE 1, inhibition of phospholipase A, with PBPB, DEX, or QUIN, alone or in combination, blocked arachidonate release by 50-60% but had no effect on EDRF production, as assessed by cyclic GMP accumulation in mixed or cocultures of endothelial and vascular smooth muscle cells. Inhibition of the phospholipase C-diacylglycerol (DAG) lipase pathway of arachidonate release by the DAG lipase inhibitor RHC-80267 also caused partial inhibition of arachidonate release and had no effect on EDRF production. When both phospholipase Az and phospholipase C pathways for arachidonate mobilization were inhibited (DEX + RHC 80267), arachidonate release was >90% inhibited. while EDRF release remained intact. We conclude