Shoichiro Takata

Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells

Specific binding sites for synthetic porcine endothelin (pET), a novel potent vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells, and its effects on cytosolic free Ca2+ concentrations ([Ca2+]i) and phosphatidylinositol (PI) response were studied in cultured rat aortic vascular smooth muscle cells (VSMC). Binding of 125I-labeled-pET to rat VSMC was time- and temperature-dependent and the cell-bound 125I-labeled-pET was resistant to dissociate. Scatchard analysis of binding studies indicated the presence of a single class of high-affinity binding sites: the apparent Kd was 2-4 X 10(-10) M and the maximal binding capacity was 11,000-13,000 sites/cell. The binding was highly specific for pET because neither well-recognized vasoconstrictors, peptide neurotoxins, nor Ca2+-channel blockers affected the binding. pET dose-dependently (10(-9)-10(-7) M) induced a transient and sustained increase in [Ca2+]i in fura-2-loaded cells of which effect was largely dependent on extracellular Ca2+, whereas it had no significant effect on PI response in 3H-myoinositol-prelabeled cells. The present data clearly demonstrates the presence of specific receptors for pET distinct from those of the well-recognized vasoconstrictors and voltage-dependent Ca2+-channels in cultured rat VSMC, and suggest that pET-induced increase in [Ca2+]i is involved in the mechanism of its vasoconstriction.

Calcitonin gene-related peptide receptor in cultured vascular smooth muscle and endothelial cells

Using 125I-labeled-Tyr0-rat(r)-calcitonin gene-related peptide (CGRP), a potent vasodilatory neuropeptide, we have identified and characterized specific binding sites for CGRP in cultured rat vascular smooth muscle cells (VSMC) and bovine endothelial cells (EC). rCGRP and human (h) CGRP equipotently inhibited 125I-rCGRP binding to both cells, but human calcitonin (hCT) was less potent and other unrelated polypeptides were ineffective. Both rCGRP and hCGRP, but not hCT, equally stimulated intracellular cAMP generation in both cells distinct from beta-adrenergic receptor-mediated mechanism, although they had no effect on cGMP generation in either cell or synthesis of prostacyclin in EC. Autoradiograph of affinity-labeled cell membranes revealed that 125I-rCGRP interacts with a single binding component of almost identical molecular size (approximately 60-kDa) in both cells under reducing and nonreducing conditions. The present study demonstrates for the first time the presence of CGRP receptors in cultured VSMC and EC, functionally coupled to adenylate cyclase system distinct from beta-adrenergic receptors. It is suggested that CGRP-induced vasorelaxation may be mediated partly by cAMP-dependent and/or endothelium-dependent mechanism.

Functional receptors for vasoactive intestinal peptide in cultured vascular smooth muscle cells from rat aorta

Specific binding sites for vasoactive intestinal peptide (VIP), a potent vasodilatory polypeptide, and its effect on formation of intracellular cyclic AMP levels were studied in cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding of 125I-labeled-VIP to cultured VSMCs was time- and temperature-dependent. Scatchard analysis of binding studies suggested the presence of two classes of high and low affinity binding sites for VIP; the apparent Kd and the number of maximal binding capacity were approximately 8 X 10(-9) M and 60,000 sites/cell (high-affinity sites) and approximately 4 X 10(-8) M and 140,000 sites/cell (low-affinity sites), respectively. Unlabeled VIP competitively inhibited the binding of 125I-labeled-VIP to its binding sites, whereas neither peptides structurally related to VIP, nor other vasoactive substances affected the binding. VIP stimulated formation of intracellular cyclic AMP in cultured VSMCs in a dose-dependent manner; the stimulatory effect of VIP on cyclic AMP formation was not blocked by propranolol and was additive with isoproterenol. The present study first demonstrates the presence of specific receptors for VIP in VSMCs functionally coupled to adenylate cyclase system. It is suggested that VIP exerts its vasodilatory effect through its specific receptors distinct from beta-adrenergic receptors.

Effects of steroid and thyroid hormones on synthesis of atrial natriuretic peptide by cultured atrial myocytes of rat

The in vitro effects of various steroid and thyroid hormones on synthesis of rat atrial natriuretic peptide (rANP) were studied using new-born rat atrial myocytes in culture. Dexamethasone, testosterone and triiodothyronine markedly stimulated both synthesis and secretion of immunoreactive (IR)-rANP with the same peak after 4-day-culture. Dexamethasone and testosterone dose-dependently (10(-7)-10(-6) M) stimulated synthesis of IR-rANP and were the most potent among various steroids tested. Triiodothyronine (T3) also stimulated synthesis of IR-rANP in a dose-dependent manner (10(-8)-10(-7) M), of which effect was more potent than that of tetraiodothyronine, whereas reverse T3 was ineffective. The present study clearly shows that glucocorticoids, androgens and thyroid hormones directly stimulate synthesis of ANP by atrial myocytes and suggests that ANP may play a potential role in mediating and/or modulating the biological effects by these hormones in the cardiovascular system.

Binding, internalization, and degradation of atrial natriuretic peptide in cultured vascular smooth muscle cells of rat

Binding, internalization, and degradation of 125I-labeled-rat atrial natriuretic peptide (rANP) were studied in cultured rat aortic vascular smooth muscle cells (VSMC). At 37 degrees C, 125I-labeled-rANP rapidly bound to VSMCs, but the cell-bound radioactivity rapidly decreased upon subsequent incubation, while the binding was slow at 4 degrees C, reaching to an apparent equilibrium after 6 hrs. The cell-bound 125I-labeled-rANP at 37 degrees C is rapidly dissociated from VSMC (t 1/2: approximately 40 min) with the appearance of degradaded product(s) of radioligand in the medium, whereas the degradation was minimal at 4 degrees C. This degradative process was blocked by inhibitors of metabolic energy production (azide, dinitrophenol), inhibitors of lysosomal cathepsins (leupeptin, pepstatin), and lysosomotropic agents (NH4Cl, chloroquine, lidocaine, methylamine, dansylcadaverine), but not by inhibitors of serine or thiol proteases. 125I-labeled-rANP initially bound to the cell-surface was rapidly internalized, and delivered to lysosomal structures, which was confirmed by autoradiographic studies. These data indicate that rANP, after binding to the cell-surface receptors, is rapidly internalized into the cells through receptor-mediated endocytosis, and subsequently degradaded by lysosomal hydrolases.

Autocrine effect of endothelin on DNA synthesis in human vascular endothelial cells

To elucidate the role of endogenous endothelin on DNA synthesis in endothelial cells, we have investigated the effects of rabbit anti-ET gamma globulin on DNA synthesis in cultured human vascular endothelial cells. DNA synthesis was markedly inhibited by anti-ET gamma globulin in a concentration dependent manner in the presence of fetal calf serum(FCS) (x5000;34%, x2500;54%, x1000;70%), but not in the absence of FCS. These data suggest that endogenous ET modulates FCS-stimulated DNA synthesis in an autocrine fashion.

Down-regulation of atrial natriuretic peptide receptor and cyclic GMP response in cultured rat vascular smooth muscle cells

Treatment of cultured rat vascular smooth muscle cells with human atrial natriuretic peptide (hANP) or Met(O)12hANP caused a similar and marked reduction (approximately 80%) of ANP receptor number (down-regulation). A second challenge with hANP stimulated the accumulation of intracellular cGMP in the down-regulated cells to the same extent as in control cells. These data suggest that ANP receptor sites are functionally heterogenous, the more abundant site being uncoupled from guanylate cyclase but susceptible to down-regulation.

Effects of protein kinase inhibitors on growth factor-stimulated DNA synthesis in cultured rat vascular smooth muscle cells

The effects of H-7 and ML-9, inhibitors of protein kinase C and myosin light-chain kinase, respectively, on DNA synthesis stimulated by platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) were studied in cultured rat vascular smooth muscle cells (VSMC). H-7 and ML-9 significantly inhibited PDGF-stimulated DNA synthesis in lower concentrations, while both compounds were only effective in inhibiting EGF-induced DNA synthesis in higher concentrations. These data suggest that protein kinase C and myosin light-chain kinase activated by PDGF play a more important role in cell proliferation of VSMC than EGF.

Regulation of atrial natriuretic peptide receptors in cultured vascular smooth muscle cells of rat

Abstract To elucidate the regulation of vascular receptors for atrial natriuretic peptide (ANP), we have studied the binding capacity of 125I-labeled rat (r) ANP using cultured vascular smooth muscle cells from rat aorta. After preincubation with 3.2×10−8M rANP at 37°C, the binding capacity decreased as a function of time; the maximal receptor loss (70–75%) occurred after 4 hrs and persisted for 24 hrs. Pretreatment with cycloheximide (20 μg/ml) and actinomycin D (2 μg/ml) similarly caused a dramatic reduction (≈80%) of the binding capacity after 24 hrs; the half-life ( t 1 2 ) of the receptor loss was ≈7–8 hrs. Following removal of rANP, the “down-regulated” ANP receptors fully recovered in the presence of 10% fetal calf serum, but not in combination with either actinomycin D or cycloheximide. Concanavalin A dose-dependently inhibited the binding. The binding capacity also decreased with time in the presence of tunicamycin (1 μg/ml) with t 1 2 of ≈30 hrs. These data indicate that protein and carbohydrate moieties are essential for the functional integrity of the vascular receptor binding sites for ANP, and suggest that the recovery of the receptor loss by “down-regulation” requires concomitant RNA and protein synthesis.

Ventricular myocytes from neonatal rats are more responsive to dexamethasone than atrial myocytes in synthesis of atrial natriuretic peptide

Using the primary culture of neonatal rat ventricular myocytes, synthesis and secretion of rat atrial natriuretic peptide (rANP) were studied. Ventricular myocytes in culture, although contained less amounts of cellular immunoreactive (IR)-rANP, secreted substantial amounts of IR-rANP at a rate comparable to that of atrial myocytes. Dexamethasone markedly stimulated synthesis and secretion of IR-rANP by cultured ventricular myocytes in a dose-dependent manner (10(-10)-10(-6) M), of which effect was far more potent than that in atrial myocytes. Testosterone and triiodothyronine also stimulated synthesis and secretion of ventricular IR-rANP to the extent comparable to that of atrial IR-rANP. The present study suggests that tissue-dependent difference in glucocorticoids sensitivity plays an important role in the regulation of developmental ANP gene expression in mammalian heart.