Desensitisation of calcitonin gene-related peptide responsiveness but not adrenomedullin responsiveness in vascular smooth muscle cells

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) are distantly related peptides. Both act through G protein-coupled receptors on vascular smooth muscle cells to increase intracellular cAMP concentrations, causing vasorelaxation. Recent evidence suggests that both peptides bind to a common heptahelical receptor, with specificity for each peptide being determined by a receptor activity modifying protein (RAMP). This hypothesis predicts that each peptide should desensitise the cellular response to subsequent stimulation by the other. We have studied the patterns of desensitisation of ADM/CGRP receptors in rat aortic vascular smooth muscle cells. Cells were incubated for 20 min in either serum free medium (SFM), alone (control) or in SFM containing vasoactive agonist (e.g. ADM 10(-8) M, CGRP 10(-7) M, angiotensin II 10(-9) M or isoproterenol 10(-6) M). Cells were then washed and incubated for a further 20 min in SFM containing a second agonist and 1 mM isobutyryl methyl xanthine. Cells were harvested and assayed for cAMP. Pre-exposure of cells to CGRP, isoproterenol, angiotensin II or ADM, decreased cAMP generation in response to subsequent stimulation with CGRP by 84% (+/-5), 66% (+/-18), 45% (+/-5) and 60% (+/-10) respectively (mean+/-s.d.). Pre-incubation of cells with 100 nM H-89, a protein kinase A (PKA) inhibitor, abolished the desensitisation of CGRP by itself, implying that this desensitisation was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with ADM by pre-exposure to ADM and all other agonists tested. Identical results were seen with or without PKA inhibition by H-89. These results indicate that the ADM receptor does not desensitise over this time period in RAVSMCs, in contrast to the CGRP receptor, which is desensitised by pre-exposure to CGRP and other vaso-active agonists. These data also suggest that ADM and CGRP act through separate receptors in these cells.     

Inhibitory effect of calcitonin gene-related peptide and calcitonin on opossum esophageal smooth muscle

Calcitonin gene-related peptide (CGRP) is widely distributed in the gastrointestinal nerves, including those of the esophagus. The present investigation was undertaken to examine the effect and the mechanism of action of CGRP on the lower esophageal sphincter and esophageal contractions. This peptide caused dose-dependent relaxation of the lower esophageal sphincter. The D50 for inhibitory effect of intraarterial CGRP on the sphincter was 5.0 X 10(-13) mol/kg. Calcitonin gene-related peptide is 3000 times more potent than calcitonin. The effect of CGRP on the lower esophageal sphincter was partially antagonized by tetrodotoxin or black widow spider venom. The inhibitory effect of CGRP on the sphincter appears to be exerted at two levels: (a) at the sphincteric smooth muscle, and (b) at the noncholinergic, nonadrenergic inhibitory neurons. Calcitonin gene-related peptide also exerts a potent inhibitory effect on the peristaltic contraction of the esophageal body in response to swallowing and vagal efferent stimulation. Using immunohistochemical studies we also showed the presence of CGRP-immunoreactive neurons within the myenteric ganglia of the esophagus. These studies suggest that CGRP may play an important role as an inhibitory neurotransmitter in the esophagus.     

Calcitonin gene-related peptide activates the K+ channels of vascular smooth muscle cells via adenylate cyclase

The aim of the present study was to examine the effects of calcitonin gene-related peptide (CGRP) on the K⁺ channels of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 nM CGRP activated two types of K⁺ channels the Ca²⁺-activated K⁺ channel (K_Ca channel) and the ATP-sensitive K⁺ channel (K_ATP channel) in cell-attached patch configurations. In cells pretreated with Rp-cAMPS, a membrane-permeable inhibitor of cAMP-dependent protein kinase (PKA), extracellular application of 100 nM CGRP could not activate the K_Ca or K_ATP channel, indicating that the activation of the K⁺ channels by CGRP occurs in connection with PKA. In the cell-attached patch configurations, extracellular application of 1 mM dibutyryl cAMP, a membrane permeable cAMP, activated K_Ca and K_ATP channels. In inside-out patch configurations, application of PKA to the cytosolic side activated both the K_Ca and K_ATP channels. These results indicate that CGRP modulates the K⁺ channels of vascular smooth muscle cells via adenylate cyclase, i.e., cAMP-PKA pathway, and contributes to control of vascular tone.     

Stimulation and homologous desensitization of calcitonin gene-related peptide receptors in cultured beating rat heart cells

Addition of calcitonin gene-related peptide (CGRP), 1 X 10(-7) M, to cultured neonatal rat heart cells resulted in rapid increases in beating rate and cellular concentrations of cAMP. Calcitonin (1 X 10(-7) M), in contrast, had no significant effect on heart cell beating rate or cAMP content. CGRP-stimulated increases in heart cell cAMP content were rapid, transient, dose dependent, and potentiated by isobutyl-methylxanthine (1 X 10(-4) M). Half-maximal increases in heart cell cAMP content occurred at 1 X 10(-8) M CGRP. Heart cell adenylate cyclase responses to CGRP were desensitized in a rapid (i.e. within 5 min) and dose-dependent manner by prior exposure to CGRP. Complete and half-maximal desensitization of heart cells to CGRP occurred at 1 X 10(-8) and 3 X 10(-10) M CGRP, respectively. Desensitization of heart cells to CGRP did not modify the cAMP response of heart cells to beta-adrenergic agonist stimulation, and beta-adrenergic agonist desensitization of heart cells did not modify responses to CGRP. Heart cell cAMP responses to CGRP were additive to those of the beta-adrenergic agonist isoproterenol and occurred in the presence of beta-adrenergic blockade. These observations demonstrate that CGRP exerts specific and potent agonist actions in cardiac myocytes and that regulation of myocardial responses to CGRP may occur by mechanisms involving increases in cAMP and receptor desensitization.     

Calcitonin gene-related peptide receptor activation by receptor activity-modifying protein-1 gene transfer to vascular smooth muscle cells

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator that plays a protective role in the cardiovascular system. The receptor for CGRP is an unusual complex of the G protein-coupled calcitonin-like receptor and an obligate receptor activity modifying protein-1 (RAMP1). In this report we provide the first evidence that RAMP1 is rate limiting in vascular smooth muscle cells. Although cultured rat aorta smooth muscle cells express calcitonin like-receptor and RAMP1, we found that CGRP is not a potent activator of the receptor. After overexpression of RAMP1 by adenoviral gene transfer, there was a striking increase in CGRP-induced production of cAMP, with a 75-fold decrease in the EC(50) and a 1.5-fold increase in the maximal response. The biological consequence of this increased receptor activity was observed in three different paradigms. First, RAMP1 gene transfer caused a CGRP-dependent decrease in cell proliferation. Second, RAMP1 and CGRP treatment led to a 3-fold greater free radical-induced reduction in cell number. Finally, RAMP1 gene transfer resulted in a 5-fold CGRP-dependent increase in terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive apoptotic cells upon serum withdrawal. The mechanisms underlying these effects involved cAMP-dependent pathways. We propose that RAMP1 gene transfer may be an effective strategy for increasing the effectiveness of CGRP-induced decrease in restenosis after aortic angioplasty.     

降钙素基因相关肽抑制原发性高血压患者动脉血管平滑肌细胞的增殖作用

目的 :探讨降钙素基因相关肽 (CGRP)对原发性高血压 (EH)患者动脉血管平滑肌细胞 (VSMC)增殖的抑制作用。方法 :对EH患者和血压正常者的动脉进行培养 ,分别测定两组VSMC的周期和数量 ,并观察CGRP对其影响。结果 :①基础状态下 ,EH组VSMC周期的S期百分率和增殖指数 (PI)分别为 (35 .78± 1.0 0 ) %和 (5 4 .84± 1.98) % ,均显著高于正常对照组的 (2 8.88± 1.0 7) %和 (4 4 .12± 1.4 3) % ,P <0 .0 1;EH组VSME数量为 (10 .39± 1.30 )× 10 9个 /L ,显著高于正常对照组的 (7.6 7± 0 .98)× 10 9个 /L ,P <0 .0 1。②在CGRP作用下 ,EH组VSMC细胞周期中的S期百分率和PI分别为 (32 .4± 1.4 7) %和 (4 8.86± 2 .2 1) % ,均显著高于正常对照组的 (2 0 .81± 0 .94 ) %和 (33.18± 1.2 6 ) % ,P <0 .0 1;EH组细胞数量为 (7.4 8± 0 .2 2 )× 10 9个 /L ,显著高于正常对照组的 (5 .37± 0 .6 6 )× 10 9个 /L ,P <0 .0 1。③在CGRP作用下 ,两组VSMC的细胞周期中S期百分率、PI和细胞数量分别较基础状态下减低非常显著 ,P <0 .0 1。结论 :EH患者VSMC的细胞数量和细胞周期中的S期百分率、PI增加 ;CGRP使VSMC的数量和PI减低 ,抑制VSMC增殖。提示CGRP含量不足可能是EH发病的机制之一     

Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells

To investigate the role of vasoconstrictor hormones in vascular smooth muscle cell growth we have studied the effects of the potent vasoconstrictor angiotensin II on cell growth in a cultured rat aortic cell model. Angiotensin II was not mitogenic for these cells, as assessed by determining cell number, nor was it synergistic in this regard with 10% calf serum. However, 24-hour exposure to 100 nM angiotensin II caused an 80% increase in protein synthesis (compared with 0.4% increase with serum control) as measured by tritiated leucine incorporation. This was a "hypertrophic" response as indicated by a 30% increase in protein content and a 45% increase in cell volume. Angiotensin II-induced smooth muscle cell hypertrophy was maximal at 100 nM, had an ED50 of 1 nM, and was inhibited by the competitive antagonist [Sar1, Ile8]angiotensin II. The increase in protein synthesis required continuous presence of angiotensin II for 6 hours and required messenger RNA (mRNA) synthesis as suggested by complete inhibition after exposure to actinomycin D. Angiotensin II-stimulated protein synthesis was dependent on a rise in intracellular Ca2+ concentration evidenced by a 70% decrease in tritiated leucine incorporation after chelation of Ca2+ with 25 microM quin 2-AM. This treatment did not alter protein synthesis induced by 10% calf serum. Decreasing extracellular Na+ to prevent Na+/H+ exchange and intracellular alkalinization did not inhibit the angiotensin II response but decreased the 10% calf serum-stimulated protein synthesis by 35%. Downregulation of protein kinase C by 24-hour treatment with phorbol 12,13-dibutyrate did not inhibit angiotensin II-induced protein synthesis, while phorbol 12-myristate 13-acetate-stimulated protein synthesis was abolished. These findings suggest that angiotensin II-induced hypertrophy, acting via a Ca2+ mechanism, may play an important role in abnormal vascular smooth muscle cell growth in certain forms of hypertension.     

Cadmium effect on the Na,K-ATPase system in cultured vascular smooth muscle cells

The present study focuses on the interaction between cadmium (Cd) and the Na, K-ATPase system in in vitro grown vascular smooth muscle cells (VSMCs) derived from the rat carotid artery. In disrupted VSMCs rendered permeable by osmotic shock, Cd inhibited Na, K-ATPase; I50 was reached at 10(-5) M Cd. Mg-ATPase was also inhibited by Cd; I50 was attained at concentrations of 10(-4) M Cd. Cd inhibition of Na,K-ATPase in the VSMCs was noncompetitive with respect to Na, K, and ATP. Rubidium transport experiments performed with intact VSMCs demonstrated that within an incubation period of 150 minutes, a concentration of 10(-4) M Cd in the extracellular fluid exerted no acute effect on the Na-K pump. Within this time interval, intracellular Cd attained a concentration eightfold higher than the extracellular Cd concentration. Thus, it appears that under acute conditions Cd exerts its inhibitory effect on Na, K-ATPase only in disrupted VSMCs. The data further suggest that, in the VSMC, conditions under which Cd inhibits Na, K-ATPase are consistent with inhibition from the cytoplasmic side of the cell membrane.     

Localization of calcitonin gene-related peptide in human esophageal Langerhans cells

Previously undescribed calcitonin gene-related peptide-immunoreactive intraepithelial cells were seen in specimens of esophageal mucosa obtained by biopsy or surgical resection from 14 individuals. These calcitonin gene-related peptide-immunoreactive cells were sparsely seen in normal mucosa but increased markedly in esophagitis. They were inaccessible to routine histological stains, but osmication showed them as dendritic forms resembling Langerhans cells of the skin. Their cytological identity was determined with immunocytochemical tests for human antigenic markers such as Ia, HLA-DR, and OKT6 for Langerhans cells, Leu-M5 and Leu-M3 for intraepithelial macrophages, CD3 and TCR-1 for T-lymphocytes, Leu-14 for B-lymphocytes, S-100 for Merkel cells, and chromogranin for amine precursor uptake and decarboxylation cells. Double localization showed that calcitonin gene-related peptide immunoreactivity colocalized with Ia, HLA-DR, and OKT6 but not with the other markers. These studies show that intraepithelial Langerhans cells in the esophageal mucosa contain calcitonin gene-related peptide, which may serve as an immunomodulator.     

Production of calcitonin gene-related peptide from human cancer cells

Calcitonin (CT) is produced ectopically from a variety of non-thyroidal cancers. The CT genes also encode another peptide, calcitonin gene-related peptide (CGRP) which is a potent vasodilator. In the present study we have used immunochemical and chromatographic methods to demonstrate the presence and characterize the molecular forms of CGRP in cultured human cancer cells. Using two highly sensitive and specific radioimmunoassays, we have detected immunoreactive CGRP (i-CGRP) in cell extracts and cell-exposed media of cultured promyelocytic leukaemia (HL60) and bronchogenic carcinoma (BEN) cells. The mean i-CGRP content of the HL60 and BEN cell extracts was 2 and 45 pmol/g wet weight respectively. On gel filtration and high performance liquid chromatography, the immunoreactive material was found to be heterogeneous, though a major proportion co-eluted with synthetic human CGRP(1-37), suggesting structural identity with the intact CGRP molecule. Finally, we have discussed some interesting features of CT-gene peptide expression in tumour cells.     

Lysosomal composition in cultured vascular smooth muscle cells: electron probe analysis.

Spherical electron-dense organelles in the perinuclear region of cultured guinea pig aortic smooth muscle cells were identified as lysosomes by their ability to accumulate acridine orange and by cytochemical demonstration of their acid phosphatase content. The number and size of lysosomes increased in subcultured cells. The elemental composition of the lysosomes was quantitated by electron probe analysis of whole freeze-dried cells and of cryosections. In lysosomes at this stage in their development, the sulfur concentration was higher than that in the cytoplasm and the K/Na concentration ratio was similar to that in the cytoplasm.     

Regulation of renin-like enzyme in cultured human vascular smooth muscle cells

Cultured human arterial smooth muscle cells produced an immunologically specific renin-like enzyme. The renin-like enzyme in the culture medium was mostly an inactive form; the proportion of the active form in the cell was 30 to 75%. Phorbol 12-myristate 13-acetate, N'-O'-dibutylyladenosine 3', 5'-monophosphate and isoproterenol with theophylline increased the renin-like enzyme in the medium and in the cell, dose dependently. Endothelial cell growth supplement also increased the renin-like enzyme produced by cultured vascular smooth muscle cells, and heparin promoted the effects of endothelial cell growth supplement. The existence of the regulation of the renin-like enzyme produced by cultured vascular smooth muscle cells strongly suggests the existence of a local renin angiotensin system in human vascular walls.     

Angiotensin II stimulates collagen synthesis in cultured vascular smooth muscle cells

The aim of the present study was to investigate whether angiotensin II, by increasing extracellular matrix synthesis, contributed to the vascular wall thickening observed in hypertension. Thus, we examined the direct effects of angiotensin II on collagen and fibronectin synthesis in cultured rat vascular smooth muscle cells by measuring 3H-proline incorporation. Angiotensin II, in a concentration of 10 mumol/l, increased collagen synthesis in a dose-dependent manner up to 1.8-fold. This increase occurred within 24 h after the addition of angiotensin II and the time required to reach maximum stimulation was approximately 48 h. This increase was receptor-mediated and correlated with an increase in its specific messenger RNA. A closer study of the collagen increase demonstrated a relatively greater increase in type V collagen than type I or type III collagen. Fibronectin synthesis was also increased 1.5-fold with 10 mumol/l angiotensin II. These data suggest that angiotensin II induces vascular wall thickening by acting directly on smooth muscle cells and enhancing the production of extracellular matrix proteins.     

Stimulation of protein-tyrosine phosphorylation by endothelin-1 in cultured vascular smooth muscle cells.

In cultured rat aortic smooth cells, endothelin-1 induced tyrosine phosphorylation of at least five proteins with molecular masses of about 79, 77, 73, 45 and 40 kDa in dose- and time-dependent manners. Platelet-derived growth factor also induced tyrosine phosphorylation of the same set of proteins in addition to other proteins including platelet-derived growth factor receptors. This growth factor markedly stimulated DNA synthesis and an increase in cell number in this cell type, but endothelin-1 failed to stimulate these responses under the same conditions. These results demonstrate for the first time that endothelin-1 induces tyrosine phosphorylation of some proteins but suggest that these reactions are not enough to stimulate proliferation of vascular smooth muscle cells.     

Colocalization of calcitonin gene-related peptide and somatostatin in pancreatic islet cells and inhibition of insulin secretion by calcitonin gene-related peptide in the rat

Calcitonin gene-related peptide (CGRP)- and somatostatin (SRIF)-containing cells were identified by immunocytochemical techniques in pancreatic islet cells of the rat. CGRP-containing cells were found primarily in the peripheral portion of the pancreatic islets. In addition, CGRP-containing cells also contained somatostatin, which identifies the islet CGRP-containing cells as D cells. In the present study, we also tested the effect of CGRP on gastrin-releasing peptide (GRP; 10(-9) M)- or cholecystokinin (CCK-8, 10(-9) M)-stimulated release of insulin from isolated rat islets in vitro. At concentrations of 10(-8)-10(-11) M, CGRP inhibited GRP- and CCK-8-stimulated release of insulin significantly when compared with GRP or CCK-8 alone. At the lowest concentration of CGRP (10(-11) M), the inhibitory effect of CGRP on CCK-8-stimulated release of insulin was statistically significant (p less than 0.05) and exceptionally potent (65-90% inhibition). We have also found that CGRP does not stimulate the release of SRIF from isolated islet cells. These findings suggest that CGRP may play a regulatory role in the release of insulin.     

Plethysmographic evaluation of the vascular effects of human calcitonin gene-related peptide in man

Calcitonin gene-related peptide (CGRP) is a neuropeptide with potent cardiovascular effects that include positive inotropic and chronotropic actions systemic vasodilation, and hypotension in animals and in man. The mechanism of action of CGRP is still, however, not clear, and in particular it is not known whether vasodilation by CGRP occurs by changes in cutaneous or in muscular blood flow, or both. The aim of the study was, therefore, to evaluate the cutaneous and muscular blood flow, at rest and after ischemic test, induced by an IV bolus 25 micrograms human CGRP infusion in 5 healthy normotensive volunteers, using a strain gauge plethysmographic procedure with venous occlusion. Human CGRP provoked a transient but significant decrease in systolic and diastolic blood pressure, associated with tachycardia, marked flushing, a significant increase in plasma noradrenaline, adrenaline, and cyclic AMP levels, and a slight, but significant, decrease in serum total calcium. Moreover, a significant increase in the carpal cutaneous blood flow at rest was observed, with no significant change in the lower extremity muscular blood flow at rest and after ischemic test. Finally human CGRP produced a significant increase in the venous partial O2 pressure and in the hematocrit and a significant decrease in the venous partial CO2 pressure. The results of the present study confirm the acute cardiovascular and metabolic effects of CGRP. In fact, hypotension, tachycardia, flushing, and the increased cutaneous blood flow indicate a systemic vasodilation by the neuropeptide, with a secondary sympathetic response, as documented by the augmented catecholamine and cyclic AMP plasma levels.     

Effect of calcitonin and calcitonin gene-related peptide on pancreatic functions in man.

Calcitonin gene-related peptide (CGRP) has recently been identified in central and peripheral nerve fibres, including those of blood vessels supplying the exocrine pancreas, and in pancreatic islet cells. Moreover, receptors have been characterised in the same tissue. The present study examined the effects of human CGRP and of calcitonin on exocrine pancreatic secretion and on islet cell function in nine healthy volunteers. CGRP (300 ng/kg/h) caused, respectively, a 25% and 31% inhibition of caerulein stimulated trypsin and amylase output which was similar to that seen with calcitonin (300 ng/kg/h). Arginine stimulated insulin and glucagon release was unaffected by either CGRP, or calcitonin. Calcitonin gene-related peptide caused cutaneous flushing, but did not affect the pulse rate or arterial blood pressure in the doses tested. Calcitonin gene-related peptide inhibits exocrine pancreatic secretion in vivo in man, but does not affect islet cell hormone release.     

Expression of calcitonin gene-related peptide in medullary thyroid cancer.

We studied the expression of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in 18 patients with medullary thyroid cancer (MTC) in the neoplastic (primary or metastatic) tissue by immunohistochemistry and in the plasma by radioimmunoassay. CT immunoreactivity was found in 100% of the primary and metastatic MTC, CGRP was expressed in 66% of the primary tumors and in 73% of the metastases. Both the number of positive cells and the degree of staining were always higher for CT than for CGRP staining. While plasma CT concentrations were always increased in patients with metastases, 3 patients with metastases had undetectable plasma CGRP levels. A positive correlation was found between plasma CT and CGRP levels. These data indicate that CGRP is frequently expressed in MTC sections and that plasma CGRP measurement is an additional marker for MTC, although has no advantage with respect to CT measurements in monitoring the progression of the disease.