The effect of amylin and calcitonin gene-related peptide on insulin-stimulated glucose transport in the diaphragm

The two peptides calcitonin gene related peptide (CGRP) and amylin at 1 uM levels in an isolated rat diaphragm preparation inhibited insulin stimulated 2-deoxy[3H]glucose transport by 30 and 60 percent, respectively; this was the case at maximal (1 uM) and sub-maximal (0.5 mU) insulin concentrations. No effect was measured on the basal level of 2-deoxy[3H]glucose transport.     

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.     

Inhibition by forskolin of insulin-stimulated glucose transport in L6 muscle cells.

The cardioactive diterpene forskolin is a known activator of adenylate cyclase, but recently a specific interaction of this compound with the glucose transporter has been identified that results in the inhibition of glucose transport in several human and rat cell types. We have compared the sensitivity of basal and insulin-stimulated hexose transport to inhibition by forskolin in skeletal muscle cells of the L6 line. Forskolin completely inhibited both basal and insulin-stimulated hexose transport when present during the transport assay. The inhibition of basal transport was completely reversible upon removal of the diterpene. In contrast, insulin-stimulated hexose transport did not recover, and basal transport levels were attained instead. This effect of inhibiting (or reversing) the insulin-stimulated fraction of transport is a novel effect of the diterpene. Forskolin treatment also inhibited the stimulated fraction of transport when the stimulus was by 4 beta-phorbol 12,13-dibutyrate, reversing back to basal levels. Half-maximal inhibition of the above-basal insulin-stimulated transport was achieved with 35-50 microM-forskolin, and maximal inhibition with 100 microM. Forskolin did not inhibit 125I-insulin binding under conditions where it caused significant inhibition of insulin-stimulated hexose transport. Forskolin significantly elevated the cyclic AMP levels in the cells; however its inhibitory effect on the above basal, insulin-stimulated fraction of hexose transport was not mediated by cyclic AMP since: (i) 8-bromo cyclic AMP and cholera toxin did not mimic this effect of the diterpene, (ii) significant decreases in cyclic AMP levels caused by 2',3'-dideoxyadenosine in the presence of forskolin did not prevent inhibition of insulin-stimulated hexose transport, (iii) isobutylmethylxanthine did not potentiate forskolin effects on glucose transport but did potentiate the elevation in cyclic AMP, and (iv) 1,9-dideoxyforskolin, which does not activate adenylate cyclase, inhibited hexose transport analogously to forskolin. We conclude that forskolin can selectively inhibit the insulin- and phorbol ester-stimulated fraction of hexose transport under conditions where basal transport is unimpaired. The results are compatible with the suggestions that glucose transporters operating in the stimulated state (insulin or phorbol ester-stimulated) differ in their sensitivity to forskolin from transporters operating in the basal state, or, alternatively, that a forskolin-sensitive signal maintains the stimulated transport rate.     

Stimulation of calcitonin secretion in the pig by calcitonin gene-related peptide

Pig thyroid glands were surgically isolated in situ and perfused with autologous blood to which was added known concentrations of calcitonin gene-related peptide (αCGRP). When thyroids were perfused with measured concentrations of CGRP within the range of 0.6–600 nM, the secretion rate of calcitonin (CT) was stimulated while the release of T₃, T₄, and somatostatin remained unchanged. Specific binding of ¹²⁵I-CGRP to pig thyroid plasma membranes was demonstrated, and binding was inhibited by unlabelled CGRP but not by CT or by other peptides unrelated structurally to CGRP. The findings indicate that the pig thyroid gland contains plasma membrane binding sites for CGRP and that CGRP is capable of stimulating the secretion of CT.     

Effects of calcitonin gene-related peptide and interleukin 6 on myoblast differentiation

Effects of calcitonin gene-related peptide (CGRP) and interleukin 6 (IL-6) on muscle cell differentiation were studied using cultured rat myoblasts (L6 cells). Cell morphology and the amounts of the messenger RNAs (mRNAs) of myogenin and Myf-5, DNA content, creatine kinase (CK) activity, and myoglobin (Mb) content in the cultured cells were examined serially over 10 days of culture. In the presence of CGRP or IL-6, the mRNAs of myogenin and Myf-5 were expressed earlier and at a higher concentration in the treated cells than in the control cells. The ratios of CK activity to DNA content (CK/DNA) and of Mb content to DNA content (Mb/DNA) on day 10 of culture also were greater than in the control cells. Furthermore, the mRNAs of myogenin and Myf-5 in cultured cells incubated with both CGRP and IL-6 increased more rapidly than in cells cultured with CGRP or IL-6 alone, and the ratios of CK/DNA and Mb/DNA on day 10 were more than twice those in the presence of CGRP or IL-6. These findings indicate that both CGRP and IL-6 facilitate the differentiation of myoblasts and may have an additive effect.     

Axoplasmic transport of calcitonin gene-related peptide in rat peripheral nerve as function of age

Calcitonin gene-related peptide (CGRP) has been implicated in the trophic regulation of acetylcholine receptors and G₄ acetylcholinesterase at the rat neuromuscular junction. Since these latter molecules exhibit significant changes with advancing age, we examined the possibility that certain aspects of CGRP transport are also influenced by aging. Double nerve ligations and CGRP radioimmunoassay of 3-mm nerve segments permitted the assessment of the peptide's apparent transport rates in sciatic nerves from 3-, 12-, and 24-month-old Fischer 344 rats. Results confirm that CGRP is conveyed by anterograde axoplasmic transport; more importantly, they suggest that CGRP is also transported retrogradely, but in smaller amounts and at slower rates. In addition, our findings indicate that the apparent rates of CGRP transport in both directions significantly decline with advancing age. These data are consistent with the notion that changes in CGRP delivery may contribute to age-related changes in junctional acetylcholine receptors and acetylcholinesterase.Special issue dedicated to Dr. Sidney Ochs.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (C-IR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Ontogeny of calcitonin gene-related peptide and calcitonin in the rat thyroid

Summary In this immunohistochemical study, the ontogenic development of calcitonin-gene-related peptide (CGRP) in the rat thyroid was investigated and compared with that of calcitonin using the indirect-immunofluorescence method. Parafollicular cells with immunoreactivity to both CGRP and calcitonin first appeared at an early stage of gestation (days 17 and 18) in the central portion of the thyroid. Cells immunoreactive to CGRP and calcitonin had became numerous by gestational day 22. After postnatal day 7, CGRP- and calcitonin-immunoreactive (CIR) cells increased rapidly both in number and in the intensity of their fluorescence. In 14- to 90-day old rats, many intensely immunoreactive cells were distributed in the central portion of the thyroid. The cells immunoreactive to CGRP and to calcitonin had an almost identical ontogenic appearance. In 14-day-old and adult rats, C-IR cells also exhibited CGRP immunostaining, suggesting that these cells simultaneously produce and store CGRP during ontogeny.     

Inhibition of insulin-stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors

In isolated rat adipocytes, basal as well as insulin-stimulated 3-O-methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 microM), nitrobenzylthioguanosine (20 microM), nitrobenzylthioinosine (35 microM) and papaverine (130 microM). Transport kinetics in the presence of 10 microM dipyridamole revealed a significant increase in the transport Km value of 3-O-methylglucose (3.45 +/- 0.6 vs 2.36 +/- 0.29 mM in the controls) as well as a decrease in the Vmax value (4.84 +/- 0.95 vs 9.03 +/- 1.19 pmol/s per microliter lipid in the controls). Half-maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 microM). The inhibitory effect of dipyridamole (5 microM) reached its maximum within 30 s. The agent failed to affect insulin's half-maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose-inhibitable cytochalasin B binding (IC50 = 1.65 +/- 0.05 microM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.     

Localization of calcitonin and calcitonin gene-related peptide mRNAs in rat parafollicular cells by hybridocytochemistry

By use of appropriate fragments of CT DNA or a CGRP DNA and SP6 polymerase system, we produced anti-sense RNA probes labeled with biotinylated 11-UTP. The labeling and specificity of the RNA probes were confirmed using dot-blot hybridization. By use of hybridocytochemistry, CT mRNA and CGRP mRNA were localized in all parafollicular cells in control and dihydrotachysterin-pre-treated rats. We concluded that all parafollicular cells simultaneously produce both CT mRNA and CGRP mRNA, either under control conditions or after stimulation by dihydrotachysterin-induced hypercalcemia.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

Localization of calcitonin gene-related peptide in cardiomyocytes: comparison of neonatal and dedifferentiating cells to adult myocytes

The purpose of this study was to localize sites of calcitonin gene-related peptide binding in neonatal, freshly isolated and dedifferentiated adult cardiac myocytes in order to help us elucidate the mechanisms of action of this neuropeptides. Previous work has shown that treatment with calcitonin gene-related peptide results in dramatic changes in calcium transients, so we carried out multi-channel acquisitions of fluorescently labeled images to reveal where calcitonin gene-related protein and the L-type calcium channel were localized. Calcitonin gene-related protein was sparse and randomly distributed in rod-like adult cardiomyocytes, found in abundance in areas of the cell where striations were apparent and not where adhesion proteins predominated in dedifferentiating adult myocytes, and in a large perinuclear concentration, with some spreading into the cytoplasm in neonatal cells. Subsequent modeling demonstrated that calcitonin gene-related peptide and the L-type calcium channel protein were closely associated in each of the three myocyte types, suggesting that while the peptide has dramatic and different effects on intracellular calcium levels of the various cardiomyocytes, the action is probably via diverse mechanisms as a result of effects on different channels or pump proteins due to alterations in intracellular calcium concentrations.     

降钙素基因相关肽与偏头痛关系的研究

偏头痛是临床上常见的多发性疾病,主要表现为一侧或双侧头部反复发作的搏动性疼痛,可伴有恶心、呕吐、视物异常等神经症状。关于偏头痛的发病机制研究尚无定论。偏头痛的发病机理一直受到人们的关注,近年来一些研究表明,偏头痛的发作与头颅血管周围组织产生的血管活性物质,特别是与三叉神经血管系统的降钙素基因相关肽(calcitonin gene-relatedpeptide,CGRP)的含量变化关系密切。     

Potent anti-inflammatory action of calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP), but not substance P (SP), was found to inhibit edema-promoting actions of inflammatory mediators (histamine, leukotrine B4, 5-hydroxytryptamine) in vivo in the hamster cheek pouch, human skin, and rat paw. The effect of CGRP was present in the low nanomolar dose range, and it was mimicked by activation of sensory nerves with capsaicin which caused release of endogenous CGRP-like immunoreactivity (IR). The findings provide new information on the potential impact of sensory nerve activation during inflammatory processes by indicating that sensory nerves may play an anti-inflammatory role.     

降钙素基因相关肽受体组分蛋白

降钙素基因相关肽受体组分蛋白(calcitonin gene-related peptide-receptor component protein,CGRP-RCP)是降钙素基因相关肽受体的一个具有146/148个氨基酸的胞内膜周边蛋白,特异地与降钙素受体样受体(calcitonin receptor-like receptor,CRLR)相互作用并促进CGRP和肾上腺髓质素的信号跨膜转导,现认为CGRP-RCP也是G蛋白偶联受体中一个动态的调节器。CGRP-RCP的mRNA在人和鼠的几乎所有组织均可检测到,在小鼠睾丸中分布尤其明显。在哺乳动物中,CGRP-RCP与C17(酵母菌中聚合酶III的必需亚基)是直系同源蛋白,人体的CGRP-RCP能取代酵母中的C17,发挥与C17相同的生物学作用。     

Biological actions of human and rat calcitonin and calcitonin gene-related peptide

We assessed the central and peripheral biological actions of human and rat calcitonin and calcitonin gene-related peptide (CGRP). After intravenous administration, human and rat calcitonin, but neither human nor rat CGRP significantly decreased plasma calcium and phosphorus concentrations in awake, freely moving rats. After intracerebroventricular as well as after intravenous administration, human and rat calcitonin and human and rat CGRP significantly inhibited gastric acid secretion in conscious rats. Intracerebroventricular administration of rat calcitonin did not alter plasma calcium and phosphorus concentrations. Linear, partially protected CGRP and calcitonin did not exhibit any biological effects. These studies indicate that calcitonin, but not CGRP, affects calcium and phosphorus homeostasis while both peptides decrease gastric acid secretion similarly. Furthermore, these studies support the hypothesis that the calcium and phosphorus lowering effects of calcitonin are peripheral while the gastric inhibiting actions of the calcitonin and CGRP are mediated by the central nervous system.     

Hemodynamic effects of calcitonin gene-related peptide in conscious rats

The cardiovascular effects of calcitonin gene-related peptide (CGRP) were examined in conscious, unrestrained rats. Changes in mean arterial pressure, heart rate and cardiac output were continuously monitored before and after i.v. bolus injection of CGRP (0.1-5 micrograms/kg). Injection of the peptide caused dose-dependent reductions in mean arterial pressure (-24 +/- 4 mmHg), which were accompanied by marked tachycardia. Cardiac output was significantly increased after CGRP but little change was observed in stroke volume. CGRP also reduced total peripheral resistance (-46 +/- 6%). These data indicate that the hypotensive actions of CGRP are mediated through peripheral vasodilation rather than through reductions in cardiac output. Pretreatment with propranolol significantly reduced the tachycardia responses to CGRP from 81 +/- 11 beats/min to 36 +/- 4 beats/min, but did not abolish the increase in heart rate. These data suggest that CGRP produces a tachycardia through reflex increases in cardiac sympathetic tone and through possible direct positive chronotropic effects on the heart.