Uterine relaxation responses to calcitonin gene-related peptide and calcitonin gene-related peptide receptors decreased during labor in rats

OBJECTIVES: Our purpose was to investigate (1) whether uterine relaxation responses to calcitonin gene-related peptide are differentially regulated during pregnancy and labor, (2) the involvement of nitric oxide in smooth muscle relaxant action of calcitonin gene-related peptide in the rat uterus, (3) whether receptors for calcitonin gene-related peptide are expressed in rat uterus, and if so (4) whether the concentrations of these receptors are differently regulated during pregnancy and labor. STUDY DESIGN: Rats were killed on day 18 of gestation, at the time of spontaneous labor, or postpartum day 2. The uteri were removed for in vitro contractility measurements, nitric oxide production, and calcitonin gene-related peptide receptor binding assay. RESULTS: (1) Calcitonin gene-related peptide induced a dose-dependent relaxation in spontaneously contracting uterine strips from pregnant rats on day 18 of gestation; (2) the relaxation effects of calcitonin gene-related peptide on the uterus were decreased during spontaneous delivery at term and post partum compared with that during pregnancy; (3) calcitonin gene-related peptide-induced relaxation was inhibited by pretreatment of the uterine tissue with a calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide(8-37); (4) nitric oxide synthesis inhibitor (N(G)-nitro-L-arginine methyl ester) and soluble guanylate cyclase inhibitor (LY83583) significantly decreased calcitonin gene-related peptide-induced relaxation of the rat uterus during pregnancy; (5) calcitonin gene-related peptide increased the uterine nitric oxide production in pregnant rats, and this increase was obliterated in the presence of calcitonin gene-related peptide(8-37); and (6) calcitonin gene-related peptide receptors are present in rat uterus, and the concentration of these receptors dramatically increases during pregnancy and decreases during labor at term. CONCLUSIONS: Calcitonin gene-related peptide inhibits uterine spontaneous contractions in rats during pregnancy but not during labor and post partum. The inhibitory effects of calcitonin gene-related peptide on uterine contractility appear to be modulated, at least in part, by the activation of nitric oxide generation in the rat uterus. Changes in calcitonin gene-related peptide receptors could contribute to the changes in calcitonin gene-related peptide-mediated uterine relaxation during pregnancy and labor.     

The distribution of calcitonin gene-related peptide in gastric vagal circuit of rats

The influence of multiple suture lines along a vein graft for arterial repair was evaluated in a microsurgical model. Forty-five rats were divided into three groups. The femoral artery was repaired using one vein graft in group I, two sequential vein grafts in group II, and three grafts in group III. Patency rates were evaluated at 48 h and 10 days, and found to be 100% in all three groups. In the present study, patency was not affected by the number of suture lines. These results suggest that the use of multiple vein grafts for microarterial repair may be safe in difficult cases.     

The role of calcitonin gene-related peptide fragments in regulating the microcirculation in rats

Fragments of the calcitonin gene-related neuropeptide's molecule with the amino acid sequence 1-9, 10-20, 15-24, 20-29 and 30-37, were studied. The fragment 20-29 revealed the greatest biological activity: it induced a dose-dependent drop of arterial pressure in i.v. administration, enlarges arterial and venous microvessels when locally applied.     

A protective role for calcitonin gene-related peptide in water-immersion stress-induced gastric ulcers in rats

For over a century, Cincinnati, Ohio, has been at the center of the nation's efforts to control water pollution. Site and subject of PHS activities to understand, manage, and prevent pollution, Cincinnati now carries on this public health legacy as home to EPA's water pollution programs. From ante-bellum way station for primary care and the seat of early 20th century scientific contributions to vibrant center for the development of environmental health programs after World War II, the Queen City has truly provided a number of watershed developments in the history of public health.     

Congenital undescended testes in neonatal pigs and the effect of exogenous calcitonin gene-related peptide

PURPOSE: We investigated the neonatal piglet as a possible animal model for cryptorchidism and to determine whether calcitonin gene-related peptide (CGRP), which has been proposed to regulate inguinoscrotal testicular descent, could induce testicular descent in piglets with congenital cryptorchidism. MATERIALS AND METHODS: We examined 38 cryptorchid piglets to document the anatomy in 8 and to investigate the role of CGRP in 30. The 2-week-old piglets were allocated randomly to receive a mini-osmotic pump containing CGRP at various concentrations or phosphate buffered saline. The pump was inserted surgically into the ipsilateral scrotum, with the contents blinded to the surgeon. The positions of the testes, pump and anatomical landmarks were measured and photographed. The pigs were sacrificed and dissected 2 weeks later, and the positions were remeasured and photographed. The testes were examined histologically. RESULTS: The 3 variants of cryptorchidism observed were intra-abdominal in 20 cases, inguinal in 9 and lateral inguinal ectopic in 9. CGRP had no effect on intra-abdominal or ectopic testes. In contrast, for inguinal testes exogenous CGRP caused a slight but significant 10 +/- 7.9 mm. descent towards the pump in 5 cases compared to -2.9 +/- 5.8 mm. in 4 controls. CONCLUSIONS: Exogenous CGRP stimulated migration of inguinal testes that had been arrested in the line of descent while ectopic testes did not respond. These results support a role for CGRP in testicular descent and suggest that a slow release depot preparation might be useful as a possible treatment in some forms of cryptorchidism.     

Inhibitory effect of calcitonin gene-related peptide on myometrial contractility is diminished at parturition

The uterus is innervated by calcitonin gene-related peptide (CGRP) immunoreactive neurons, and CGRP inhibits spontaneous and evoked contractions in the uterus and fallopian tubes. In the present study using isometric force measurements on myometrial strips, we determined that CGRP inhibition of acetylcholine-induced contractions was drastically reduced at parturition compared with earlier stages of pregnancy in mice. The levels of inhibition exerted by CGRP paralleled the expression of a novel protein recently implicated in CGRP receptor activation, the CGRP-receptor component protein (CGRP-RCP). The mouse CGRP-RCP complementary DNA was isolated from uterus, and expression of the CGRP-RCP was monitored during gestation by Northern and Western blot analysis. Although CGRP-RCP messenger RNA levels did not vary significantly during gestation and postpartum, CGRP-RCP protein was greatly diminished at parturition. This diminution correlated with the loss of CGRP inhibition of acetylcholine-induced contractions observed in the force experiments. A role for CGRP and CGRP-RCP in modulation of myometrial smooth muscle contractility during pregnancy and in labor is suggested.     

Dimeric form of calcitonin gene-related peptide in the porcine thyroid gland

In this study we investigated peptides that increase rat platelet cAMP in porcine thyroid gland. Gel filtration of extracts from porcine thyroid gland showed high and low molecular weight activity. Low molecular weight activity contained peptides, including calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI). We isolated a high molecular weight peptide (M. W. 11,000) showing potent activity able to increase rat platelet cAMP in porcine thyroid gland. The peptide's N-terminal sequence was determined to be Ser-X-Asn-Thr-Ala-Thr- by gas phase sequencer analysis, a sequence identical to that of porcine CGRP. The peptide had CGRP immunoreactivity as well as platelet cAMP elevating activity. By gel filtration HPLC, synthetic human CGRP (M. W. 3790) was eluted in a position corresponding to M. W. 5,500. These results suggest that judging from its high molecular weight the above peptide is a dimeric form of CGRP.     

Hormonal replacement therapy affects calcitonin gene-related peptide and atrial natriuretic peptide secretion in postmenopausal women

OBJECTIVE: Menopause is associated with critical changes in the cardiovascular system, and the possible effect of hormonal replacement therapy (HRT) on these changes is under investigation. The aim of our study was to evaluate in postmenopausal women the effects of HRT and clonidine on the response of plasma calcitonin gene-related peptide (CGRP) and plasma atrial natriuretic peptide (ANP) to the upright posture test and the saline infusion test respectively. METHODS: CGRP and ANP levels were measured with specific radioimmunological assays and expressed in pmol/l (means +/- S.E.M). DESIGN: Postmenopausal women (age 46-53 years) (n = 18) were studied before and after 3 months of HRT (n = 13) or clonidine treatment (n = 5). RESULTS: After HRT or clonidine treatment plasma CGRP levels (14.9 +/- 1.6 and 15.9 +/- 3.8 pmol/l) were significantly higher than before (9.8 +/- 0.6 and 10.5 +/- 1.6 pmol/l) (P < 0.01). The assumption of upright posture caused no change in plasma CGRP levels before treatment, while after HRT, but not after clonidine treatment, an increase in plasma CGRP levels was observed (P < 0.01 at 5 and 20 min). Basal plasma ANP levels significantly decreased after both HRT and clonidine treatment (P < 0.01). In untreated women the saline infusion test did not induce any change in plasma ANP levels; a significant response to the test was restored after HRT but not after clonidine treatment (P < 0.01 at 90 and 120 min). CONCLUSIONS: The results show that some of the adaptive responses modified by menopausal changes are restored by HRT but not clonidine treatment, suggesting a modulatory role for sex steroid hormones in cardiovascular function and salt and water balance.     

Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats

1. The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin generelated peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats. 2. The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94+/-0.22 and 43.9+/-5.2, respectively. 3. This vasodilator response to insulin was unaffected by 100 nM propranolol (beta-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 microM L-NG-nitroarginine (nitric oxide synthase inhibitor), 1 microM ouabain (Na+-K+ ATPase inhibitor), or 1 microM glibenclamide (ATP sensitive K+-channel inhibitor). 4. In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62+/-0.21 and 81.0+/-4.6, respectively, significantly greater than in preparations with intact endothelium. 5. The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8 37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation. 6. These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors.     

Changes in pulmonary calcitonin gene-related peptide and protein gene product 9.5 innervation in rats infected with Mycoplasma pulmonis

Epithelial-mesenchymal interaction is a prerequisite for tooth morphogenesis. To study this interaction, inner enamel epithelium and dental papilla mesenchyme of molar tooth germs from a 16.5-day mouse embryo were dissociated enzymatically and cultured alone or after recombination. Characteristic matrix protein synthesized and secreted by recombined tooth germ was determined quantitatively by enzyme-linked immunosorbent assay. The protein was detected in the culture of recombined tooth germ but not of dissociated enamel epithelium alone. The amount of enamel protein increased until 8 days in culture. Morphological differentiation of the recombined epithelial rudiment into ameloblasts and enamel protein production were confirmed.     

Protective effect of D-glucaro-delta-lactam against amino-glycoside-induced nephrotoxicity in rats

The nephrotoxicity of rats caused by dibekacin (3',4'-dideoxykanamycin B) or kanamycin with or without dextran was effectively reduced by D-glucaro-delta-lactam potassium salt, as evidenced by lower levels of blood urea nitrogen and kidney edema rate, better excretion of antibiotics,and less morphological damage. Protection was dosage related, and potentiated with increasing doses, but only when the two drugs were given simultaneously. Among three alkali-metal salts examined, the potassium salt was almost equal to the lithium salt, but surpassed the sodium salt in effectiveness. Inorganic salts, in particular potassium chloride were found to be effective for the protection of normal rats, but their effect decreased for the dehydrated rats, especially in the presence of dextran.     

Polyamines regulate human medullary thyroid carcinoma TT-cell proliferation and secretion of calcitonin and calcitonin gene-related peptide

The significance of polyamines for the neoplastic proliferation and secretion of calcitonin (CT) and calcitonin-gene-related peptide (CGRP) by the human medullary thyroid carcinoma TT cell line was investigated. TT cells were cultured in vitro for 6 days with or without additions of pathway inhibitors of polyamine biosynthetic enzymes. Treatment of the cells with 1 mM of the specific L-ornithine decarboxylase (ODC) inhibitor DL-alpha-difluoromethylornithine (DFMO) resulted in a 97% decrease in ODC activity, lowered contents of putrescine (96%) and spermidine (85%) and cell proliferation rates (90%) along with a compensatory 15-fold increase in S-adenosyl-L-methionine decarboxylase (SAMDC) activity. DFMO treatment also led to a decrease in cellular content of CT (33%) and CGRP (26%), while the drug enhanced secretion of CT (31%) but depressed that of CGRP (26%), and elevated the ratio of CT to CGRP secreted into the medium by 74%. Ethylglyoxal bis(guanylhydrazone) (EGBG), a SAMDC inhibitor, at 100 microM evoked a similar reduction of cell proliferation and lowered the content of spermine by 81%. Furthermore, EGBG treatment caused a 34-fold increase in ODC activity and a subsequent 35-fold build-up of putrescine, but also seemed to stabilize SAMDC as evidenced by a highly enhanced SAMDC activity (approximately 200-fold) during enzyme assays in the absence of the inhibitor. EGBG exposure resulted in an increase in cellular CT content (110%) and secretion of the hormone (82%), while not affecting CGRP content or release.2+ EGBG effects were partially counteracted by DFMO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenomedullin, amylin, calcitonin gene-related peptide and their fragments are potent inhibitors of gastric acid secretion in rats

A release of radio-immunoassayable LHRH from the stalk-median eminence of neonatal piglets and prepubertal gilts was measured using an in vitro incubation system. The stalk-median eminence was collected from one-week-old male (n = 19) and female (n = 21) piglets and from 6-month-old prepubertal ovariectomized gilts given oestradiol benzoate (20 micrograms/kg b.w.; n = 52) or left untreated (control; n = 25) 30 or 68 h before slaughter. Each vial, containing the stalk-median eminence in 2 ml of Krebs-Ringer bicarbonate buffer, was incubated for 30 min, followed by 30 min incubations during which either basal release or the effect of adrenoreceptor antagonists and agonists on LHRH output was evaluated. There were no differences between the basal release of LHRH (x +/- SEM; pg/ml) from the stalk-median eminence of male (65.5 +/- 9.8) and female (66.3 +/- 9.6) newborn piglets. The addition of propranolol (10(-6) M) caused a 250% increase in LHRH release from the stalk-median eminence explants of neonatal males (p = 0.08) and females (p < 0.05). Neither norepinephrine nor phentolamine affected LHRH release from the stalk-median eminence of newborn males and females. The basal release of LHRH (pg/ml) from the stalk-median eminence explants collected from ovariectomized gilts given oestradiol benzoate 30 and 68 h before slaughter or left untreated was similar (147.5 +/- 36.1, 236.4 +/- 77.7 and 202.0 +/- 41.6, respectively). Propranolol evoked a significant increase in LHRH secretion from the stalk-median eminence in the control group, but not in the groups given oestradiol benzoate. Norepinephrine (10(-6) M) increased LHRH release from the stalk-median eminence collected from the control animals, 30 h and 68 h after oestradiol benzoate treatment by 48, 78 and 73 percent, respectively. Phentolamine (10(-6) M) did not affect LHRH release from the stalk-median eminence in control animals and ovariectomized gilts primed with oestradiol benzoate. Urapidil (10(-6) M, alpha 1-adrenoreceptor antagonist) did not affect the basal LHRH release from the stalk-median eminence of gilts from the control group and group slaughtered 30 h after oestradiol benzoate treatment, but caused a rapid increase of LHRH release from the stalk-median eminence 68 h after oestradiol benzoate treatment. Phenylephrine (10(-6) M) did not affect LHRH output from the stalk-median eminence collected at various time periods after oestradiol benzoate administration in vivo. These results suggest that in pigs, nerve terminals releasing LHRH at the stalk-median eminence level are sensitive to adrenergic stimulation or inhibition and that the adrenergic system can be modulated by estrogens in the prepubertal gilts.     

Cardiac expression of genes encoding putative adrenomedullin/calcitonin gene-related peptide receptors

Recent studies have suggested that adrenomedullin (AM) may play a role in the pathophysiology of heart disease, though the specific cardiac receptors involved have not been defined. RT-PCR cloned fragments of three putative AM/calcitonin gene-related peptide (CGRP) receptors were used to established a quantitative RNase protection assay to identify and quantitate expression of receptor mRNAs in heart and in cardiac myocytes. Intact rat heart expressed mRNA encoding the putative AM/CGRP receptors RDC1 and CRLR at 37- and 15-fold higher levels, respectively, than the AM-selective receptor L1, with a qualitatively similar profile in cultured neonatal cardiac myocytes. The high level of expression of RDC1 and CRLR suggests that both AM and CGRP may have direct actions on the cardiac myocyte via common receptors that can interact with either ligand.     

Receptors for calcitonin gene-related peptide (CGRP) in the rat adrenal cortex

We previously demonstrated the presence of adrenomedullin receptors in the rat adrenal cortex. There is evidence, however, that the actions of adrenomedullin may also be mediated by the CGRP receptor. The present study was designed to determine whether specific CGRP receptors are present in the rat adrenal cortex. Adrenal glands were, sectioned and immunostained with a primary antibody raised against the first intracellular loop of the CGRP-I receptor. Staining was visualised using alkaline phosphatase and vector red. Immunostaining for the CGRP-I receptor was found in the zona glomerulosa and the adrenal medulla, but not in the inner adrenocortical zones. ACTH treatment caused an increase in staining intensity in the glomerulosa. Ligand binding studies suggested the existence of two populations of CGRP binding sites, one with a Kd of 0.1 nM, the second of 37 nM. Only CGRP-I and adrenomedullin displaced labeled CGRP binding. These results suggest that the CGRP-I receptor is expressed in the adrenal zona glomerulosa and that a second class of binding site is also present. The CGRP-I receptor appears to be regulated by ACTH.     

Roles of calcitonin gene-related peptide (CGRP) in hyperpnea-induced constriction in guinea pigs

It has been reported that hyperpnea-induced bronchoconstriction in guinea pigs is a potential model for exercise-induced asthma in humans. We hypothesized that calcitonin gene-related peptide (CGRP) could modulate leukotriene D4 (LTD4)-induced responses and be involved in the pathophysiology in this asthma model. We measured tracheal (Ptr) and alveolar pressure (PA) using alveolar capsules in open-chested, mechanically ventilated (f = 1 Hz, VT = 9 ml/kg, PEEP = 4 cm H2O) guinea pigs. Animals were intravenously pretreated with saline (SAL), CGRP(8-37) (CGRP receptor antagonist), CGRP, MK-571 (LTD4 receptor antagonist), MK-886 (5-lipoxygenase inhibitor), or CGRP(8-37) + MK-571, and then underwent dry gas hyperpnea challenge (HC, 95% 02-5% CO2, 150 breaths/min, 7 min). We calculated resistance of lung (RL), tissue (Rti), and airway (Raw). HC increased RL, Rti, and Raw in SAL controls (322 +/- 27, 430 +/- 59, 299 +/- 23% baseline, respectively). MK-571, MK-886, and CGRP significantly reduced the responses to HC, while CGRP(8-37) enhanced HC-induced responses. Pretreatment with CGRP(8-37) and MK-571 in combination attenuated HC-induced constriction. In addition, pretreatment with CGRP reduced responses induced by intravenous administration of LTD4. These observations suggest that CGRP might be involved in the pathophysiology of hyperpnea-induced constriction in guinea pigs via modulation of LTD4-elicited responses.     

Brain natriuretic peptide enhances the endothelium-independent cAMP and vasorelaxant responses of calcitonin gene-related peptide in rat aorta

The localization of cathepsin K protein in mouse osteoclasts was examined by immunolight and immunoelectron microscopy using the avidin-biotin-peroxidase complex method with anti-cathepsin K (mouse) antibody. With light microscopy, a strong immunoreaction for cathepsin K was found extracellularly along the bone and cartilage resorption lacunae and detected intracellularly in vesicles, granules, and vacuoles throughout the cytoplasm of multinuclear osteoclasts and chondroclasts attached to the surface of the bone or cartilage. Mononuclear cells, probably preosteoclasts, some distance from the bone also contained a few cathepsin K-positive vesicles and granules. Cathepsin K was sometimes found in the cisternal spaces of the rough endoplasmic reticulum and vesicles of the Golgi apparatus with electron microscopy of the basolateral region of the osteoclasts. Cathepsin K-positive vesicles and granules as lysosomal compartments were present in various stages of fusion with vacuoles as endosomal compartments that contained fragmented cathepsin K-negative fibril-like structures. Some of the vacuoles (endolysosomes), which seemed to be formed by this process of fusion, contained cathepsin K-positive vesicles and fibril-like structures that did not show the regular cross striation of type I collagen fibrils. In the apical region of the osteoclasts, cathepsin K-positive vesicles and pits had already fused with or were in the process of fusing with the ampullar extracellular spaces. There were large deposits of cathepsin K on fragmented fibril-like structures without regular cross striation in the extracellular spaces, as well as on and between the cytoplasmic processes of the ruffled border. There were also extensive deposits of cathepsin K on the type I collagen fibrils with cross striation in the bone resorption lacunae. Osteoblasts and osteocytes were negative for cathepsin K. In the immunocytochemical controls, no immunoreaction was found in the osteoclasts or preosteoclasts, or on the collagen fibrils in the resorption lacunae. The results indicate that cathepsin K is produced in mature osteoclasts attached to the bone and secreted into the bone resorption lacunae. The findings suggest that cathepsin K participates in the extracellular degradation of collagen fibrils in the resorption lacunae and in the subsequent degradation of the fragmented fibrils in the endolysosomes. It is also suggested that cathepsin K degrades the organic cartilage matrix.     

Intraepidermal nerve fiber expression of calcitonin gene-related peptide, vasoactive intestinal peptide and substance P in psoriasis

In order to evaluate more fully the role of neuropeptides in the pathogenesis of psoriasis, skin biopsies were obtained from 36 patients with psoriasis to identify substance P (SP), vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). Lesional and nonlesional skin was examined from these biopsies and the results compared with those from biopsies taken from patients with a variety of other inflammatory dermatoses, including lichen planus, lichen simplex chronicus, spongiotic dermatitis, and seborrheic dermatitis. Also studied was a series of nine biopsies taken from patients with no known skin disorders. We found an increase in the number of SP-positive nerve fibers within the epidermis in biopsies from lesional skin of psoriasis patients (8.4 nerves per 3-mm biopsy) compared with nonlesional psoriatic skin (2.6 nerves per 3-mm biopsy) and normal skin (2.0 nerves per 3 mm biopsy). Other inflammatory disorders also demonstrated fewer SP-positive nerves than lesional psoriatic skin; lichen planus (0 nerves per 3 mm biopsy) and lichen simplex chronicus (1.3 nerves per 3 mm biopsy). The difference in SP-positive nerve expression between lesional psoriatic skin and the group comprising nonlesional skin, normal skin, lichen planus, and lichen simplex chronicus attained statistical significance (P < 0.013). SP-positive intraepidermal nerve fibers in lesional psoriatic specimens were fewer than in spongiotic dermatitis (17.4 nerves per 3 mm biopsy). There was no significant difference in numbers of VIP- or CGRP-immunopositive intraepidermal nerve fibers between psoriatic skin and the group comprising all other material tested. However, in five patients with psoriasis, there was a marked increase in the expression of intraepidermal CGRP (up to 10.7 nerves per 3-mm biopsy) and VIP (up to 8.3 nerves per 3-mm biopsy) which was not observed in control groups. These findings suggest that neuropeptides SP, CGRP, and VIP play a role in the pathogenesis of psoriasis.