Calcitonin—C.N.S. action to control the pattern of intestinal motility in rats

Synthetic calcitonin injected into the lateral ventricles (ICV) of rats at picomolar concentration restores the “fasted” motility pattern of the small intestine in fed rats at doses as low as 0.083 picomoles. This effect which appeared in less than 5 min and persisted at least 2 hours for 0.83 picomole, was blocked by a previous intraventricular administration of 10 μg of calcium gluconate. At 0.83 picomole ICV, calcitonin also suppressed the disruption of the “fasted” pattern induced by intravenous infusion of Pentagastrin (6 μg·kg^?1·h^?1) but not that induced by insulin (0.5 U·kg^?1). These findings support the hypothesis that calcitonin acts centrally to control the pattern of intestinal motility by inhibiting the digestive influences responsible for the “fed” pattern. All of these peripheral influences are mediated by a Ca⁺⁺ sensitive central structure.     

Effects of intracerebroventricular administration of neurotensin,substance P and calcitonin on gastrointestinal motility in normal and vagotomized rats

The effects of intracerebroventricular (ICV) vs. intravenous (IV) injection of neurotensin, substance P and calcitonin on intestinal myoelectrical activity were examined in fed rats. ICV administered neurotensin and calcitonin restored the ‘fasted’ pattern of intestinal activity, i.e. the migrating myoelectric complex (MMC) at a dose as low as 12 and 0.2 pmol, respectively, whereas substance P only reduced significantly ($\text{P < 0.01}$) the duration of the postprandial pattern when injected ICV (48 pmol).Administered systemically at doses 100 times higher than the smallest active doses by the ICV route, calcitonin induced a fasted pattern, while neurotensin and substance P did not modify the fed pattern.The effects of ICV administration of neurotensin and calcitonin were abolished after vagotomy but the shortening effect of substance P on the duration of the postprandial pattern was still present.It is concluded that these three neuropeptides act centrally to control the pattern of intestinal motility in fed rats by shortening the ‘fed’ pattern for substance P and by restoring the MMC pattern for calcitonin and neurotensin, this last effect being mediated by the vagus.     

Central actions of calcitonin on body temperature and intestinal motility in rats: evidence for different mediations

The effects of intracerebroventricular (i.c.v.) administration of calcitonin and PGE2 on intestinal motility and body temperature were examined in conscious rats chronically fitted with intraparietal electrodes in the small intestine, a cannula in a cerebral lateral ventricle and a subcutaneous thermistor probe. Both calcitonin and PGE2 restored the fasted pattern of intestinal motility in fed rats and induced an increase in body temperature. Indomethacin, an inhibitor of the cyclooxygenase with calcium antagonistic properties, and TMB-8, an intracellular calcium antagonist, blocked the effects of calcitonin on intestinal motility and body temperature. Piroxicam, an inhibitor of the cyclooxygenase which does not affect calcium uptake blocked the thermic but not the intestinal effects of calcitonin. TMB-8 but not indomethacin or piroxicam partially blocked the effects of PGE2 on both intestinal motility and body temperature. It is concluded that the central hyperthermic effect of calcitonin is mediated through the formation and the release of prostaglandins whereas the central action of calcitonin on digestive motility results from intracerebral effects on calcium fluxes.     

A tetrapeptide isolated from hamster embryo with central opiate properties on gastrointestinal motility but not on pain perception

The effects of centrally administered kentsin (H-Thr-Pro-Arg-Lys-OH) on intestinal motility and on pain perception were investigated in rats chronically equipped with lateral ventricle catheters. Intestinal motility was recorded electromyographically from electrodes placed on the duodeno-jejunum; analgesia was evaluated by the hot-plate and tail-flick tests. Kentsin (4.0 ug/kg), injected intracerebroventricularly (ICV) 2 hours after the beginning of a meal, restores the "fasted" i.e. the migrating myoelectric complex of intestinal motility, while a 5 times higher dose administered subcutaneously was inactive. The ICV effect of kentsin was blocked by previous ICV administration of naloxone (400 ug/kg). In contrast, kentsin administered ICV (40 ug/kg) or SC (200 ug/kg) did not affect significantly (P greater than 0.05) the time latency in the two analgesic tests during 90 minutes after its administration and did not significantly modify the analgesic effects of (D5-Ala2, Met5) enkephalinamide. We conclude that kentsin when centrally administered acts on opiate receptors to alter gastrointestinal motility but without effects on pain perception.     

Involvement of dopamine in the central effect of neurotensin on intestinal motility in rats

The effects of intracerebroventricular (ICV) administration of neurotensin (NT) before a meal on intestinal postprandial motility were examined in conscious rats chronically fitted with intraparietal Nichrome electrodes in the duodeno-jejunum. The effects were compared with those of two analogues, [D-Tyr¹¹]NT and [D-Trp¹¹]NT, resistant to degradation by brain peptidases. NT (10 μg ICV) delayed the occurrence of postprandial disruption of duodenal motility and blocked it on the jejunum. [D-Tyr¹¹]NT and [D-Trp¹¹]NT (1 μg ICV) elicited the same effects but at a ten-fold lower dose. NT administered peripherally just before a meal significantly lengthened the duration of the postprandial motor pattern. The central effect of NT on the fed pattern involved dopaminergic neurons as it was mimicked by dopamine, blocked by haloperidol and partly antagonized by either sulpiride or (+) SCH 23390. It is concluded that: 1) both D₁ and D₂ receptors are involved in the blocking effect of the postprandial disruption induced by central NT; 2) that [D-Tyr¹¹]NT and [D-Trp¹¹]NT are potent agonists at NT receptors in the brain.     

Central effects of growth hormone-releasing factor (GRF) on intestinal motility in dogs: involvement of dopaminergic receptors

The effects of intracerebroventricular (ICV) and intravenous (IV) administration of human pancreatic growth hormone-releasing factor (hpGRF) on gastro-intestinal motility were examined in fasted and fed conscious dogs equipped with chronically implanted strain-gauges on the antrum and the jejunum. During the fasted state, hpGRF injected ICV at 0.1 micrograms . kg-1 or IV at 0.5 micrograms . kg-1 did not affect the cyclic occurrence of the migrating motor complex (MMC). This pattern was normally disrupted for 8-10 hours by a daily standard meal. Injected ventricularly (0.1 micrograms . kg-1) but not intravenously (0.5 micrograms . kg-1) 10-15 min after the daily meal, hpGRF significantly reduced (p less than 0.01) the duration of the jejunal fed pattern (2.0 +/- 1.4 vs. 8.4 +/- 1.1 hours for control) but not that of the stomach. This effect persisted when hpGRF (0.1 micrograms . kg-1 ICV) was administered after indomethacin (2 mg . kg-1 IM), naltrexone (0.1 mg . kg-1 IV) or domperidone (1 mg . kg-1 IV) but was abolished by a previous IV injection of metoclopramide (1 mg . kg-1). It was concluded that hpGRF is able to act centrally to control the pattern of jejunal motility in fed but not in fasted dog, its effect being probably mediated through dopaminergic pathways.     

Intraventricular calcitonin gene-related peptide inhibits gastric acid secretion

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide recently demonstrated to be a peptide expressed by the calcitonin gene in the rat central nervous system. Intracerebroventricular administration of CGRP in pylorus ligated rats resulted in a dose dependent suppression of gastric acid secretion. This effect was also present in acutely vagotomized rats. In addition, CGRP inhibited the stimulation of gastric acid secretion by thyrotropin releasing hormone. CGRP was considerably less potent in its effect on gastric acid than calcitonin, a well known central inhibitor of gastric acid secretion in the rat. This study suggests that CGRP may be a factor in the central regulation of gastric acid secretion in the rat.     

Distribution of Calcitonin Gene-Related Peptide in Rat and Rabbit Spinal Cords: Effect of Intrathecal 5,7-Dihydroxytryptamine

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was measured in selected regions of the cervical, thoracic, and lumbar spinal cord of untreated rabbits and, following intrathecal injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), in the thoracolumbar cord in rats using a sheep antiserum raised against tyrosine0 calcitonin gene-related peptide28-37. In the cervical, thoracic, and lumbar segments of the rabbit spinal cord, CGRP-LI levels were 15-50-fold higher in the dorsal than in the ventral grey region in the same segment. The only segmental variation in CGRP-LI levels was in the dorsal white region, where levels in the thoracic cord were lower than those in cervical or lumbar segments. Within individual spinal segments, the pattern of distribution of CGRP-LI in the rabbit spinal cord was analogous to that in other species previously examined, including rat, human, and cat spinal cord. Intrathecal injection of 5,7-DHT, which caused 85-91% depletion of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid from the thoracolumbar ventral spinal cord, did not affect choline acetyltransferase activity, which is colocalized with CGRP in motoneurones in this spinal cord region. In contrast, intrathecal 5,7-DHT produced a threefold increase in CGRP-LI in the ventral thoracolumbar cord, suggesting that spinal motoneurones selectively increase production of CGRP 10 days after neurotoxin-induced denervation of bulbospinal raphe neuronal input.     

Effects of corticotropin-releasing factor, corticotropin and cortisol on gastrointestinal motility in dogs

Gastrointestinal motor activity following intracerebroventricular (ICV) and intravenous (IV) administration of corticotropin releasing factor (CRF), corticotropin (ACTH) and cortisol was investigated in fasted dogs with strain-gauge transducers chronically implanted on the antrum and proximal jejunum. ICV but not IV administration of CRF (20 to 100 ng/kg) suppressed the gastric cyclic migrating motor complex (MMC) for 3 to 6 hours without affecting the jejunum. Similar disruptive effects on the gastric MMC were observed after ICV administration of ACTH (0.5 U/kg) or cortisol (0.1 micrograms/kg) but not after IV administration of 10 times higher doses. These results suggest that in dog CRF may be involved in the central control of the interdigestive gastric motility, these effects were not probably due to the release of ACTH and cortisol the other hormones of the pituitary adrenocortical system change the gastric motility when centrally administered through a possible feed-back mechanism affecting brain CRF level.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

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.     

Peripheral and Central Administration of Xenin and Neurotensin Suppress Food Intake in Rodents

Xenin is a 25‐amino acid peptide highly homologous to neurotensin. Xenin and neurotensin are reported to have similar biological effects. Both reduce food intake when administered centrally to fasted rats. We aimed to clarify and compare the effects of these peptides on food intake and behavior. We confirm that intracerebroventricular (ICV) administration of xenin or neurotensin reduces food intake in fasted rats, and demonstrate that both reduce food intake in satiated rats during the dark phase. Xenin reduced food intake more potently than neurotensin following ICV administration. ICV injection of either peptide in the dark phase increased resting behavior. Xenin and neurotensin stimulated the release of corticotrophin‐releasing hormone (CRH) from ex vivo hypothalamic explants, and administration of α‐helical CRH attenuated their effects on food intake. Intraperitoneal (IP) administration of xenin or neurotensin acutely reduced food intake in fasted mice and ad libitum fed mice in the dark phase. However, chronic continuous or twice daily peripheral administration of xenin or neurotensin to mice had no significant effect on daily food intake or body weight. These studies confirm that ICV xenin or neurotensin can acutely reduce food intake and demonstrate that peripheral administration of xenin and neurotensin also reduces food intake. This may be partly mediated by changes in hypothalamic CRH release. The lack of chronic effects on body weight observed in our experiments suggests that xenin and neurotensin are unlikely to be useful as obesity therapies.     

Calcitonin gene-related peptide-mediated cardioprotection of postconditioning in isolated rat hearts

Previous studies have demonstrated that endogenous calcitonin gene-related peptide (CGRP) plays an important role in mediation of ischemic preconditioning. In the present study, we tested whether CGRP is also involved in mediation of the protective effects of postconditioning in isolated rat hearts. Sixty minutes of left coronary artery occlusion and followed by 60 min of reperfusion caused a significant decrease in cardiac function and a significant increase in creatine kinase (CK) release and infarct size. Postconditioning with three cycles of 1-min ischemia and 1-min reperfusion produced a marked improvement of cardiac function and decreased CK release and infarct size, concomitantly with an increase in the release of CGRP release in coronary effluent. However, the cardioprotection afforded by postconditioning was abolished by CGRP 8-37 (10^− 7 M), a selective CGRP receptor antagonist, or pretreatment with capsaicin (50 mg/kg, s.c.), which depletes transmitters in sensory nerves. Exogenous CGRP (5 × 10^− 9 M) administration of CGRP reappeared postconditioning-like cardioprotection in the rats pretreated with capsaicin. These results suggest that the protective effects of ischemic postconditioning are related to stimulation of endogenous CGRP release in rat hearts.     

坐骨神经结扎后大鼠背根神经节和脊髓CGRP表达的变化

目的研究大鼠坐骨神经结扎后降钙素基因相关肽(calcitoningene-relatedpeptide,CGRP)表达变化。方法SD大鼠随机分为假手术对照组和坐骨神经结扎组,实验组结扎后分别存活1、3、5、7、14、21和28d(n=8),免疫荧光(双标法)和免疫组织化学(SABC法)观察术后不同时间点CGRP和NGF在坐骨神经、背根神经节(dorsalrootganglion,DRG)和脊髓的表达变化,Westernblot结合图像分析技术对不同时间的变化进行定量测定。结果术后1d结扎远端坐骨神经内NGF大量堆积,持续到28d仍高于正常。结扎后7dDRG内CGRP阳性细胞百分率减少,持续到28d仍低于正常;结扎后14d脊髓后角CGRP下降,28d仍低于正常,各时间点脊髓前角CGRP表达未见明显变化。结论神经结扎可导致DRG和脊髓后角的CGRP表达下调,可能与靶源性的NGF来源减少有关。     

Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system

Using an antiserum directed against human calcitonin gene-related peptide (hCGRP), which fully cross reacts with rat CGRP, a sensitive radioimmunoassay was developed. The antiserum was characterized by displacement curve characteristics and high performance liquid chromatography. The assay was applied to rat brain tissue and the concentration of CGRP for 48 microdissected brain areas is presented. Highest levels (1000–4500 fmol/mg protein) were found in the central amygdaloid, caudate putamen, and spinal trigeminal nerve nucleus and tract, substantia gelatinosa, and the dorsal horn of the spinal cord. Moderate levels (200–600 fmol/mg protein) were found in the bed nucleus of the stria terminalis, the subfornical organ, the paraventricular, arcuate, dorsomedial, dorsal parabrachial, ambiguus and tractus solitarii nuclei and in the median eminence. These results coincide with those previously obtained by immunohistochemistry. The widespread distribution in the brain suggests involvement of CGRP in a variety of behavioral functions.     

Electro-acupuncture upregulates CGRP expression after rat spinal cord transection

Calcitonin gene-related peptide (CGRP) plays a variety of important roles within the nervous system. Increasing CGRP expression could improve the survival of injured neurons and prevent neuronal loss. In this study, we first evaluated in vitro the neuroprotective function of CGRP on mechanically injured cerebellar granule neurons (CGNs) of rats. We then verified this result through exogenous administration of CGRP in a spinal cord transected completely in rats. Finally, we investigated the effect of electro-acupuncture (EA) on CGRP expression following the spinal cord transected completely in rats. We found that EA can improve CGRP expression, and exogenous CGRP may promote the survival of injured neurons, both in vivo and in vitro. Our results suggest that CGRP may be a specific neuropeptide expressed in GV-EA treatment of spinal cord injuries (SCI), and that CGRP may play a neuroprotective role in survival of neurons injured mechanically.     

Calcitonin and calcitonin gene-related peptide alter the excitability of neurons in rat forebrain

Individual neurons in the hypothalamus, thalamus, cortex, and other forebrain areas of urethane-anesthetized, male rats were iontophoretically tested for their membrane sensitivity to salmon calcitonin (CT), human CT, and CT gene-related peptide (CGRP). Extracellular recording of unit activity revealed that depression of neuronal firing was the predominant effect of iontophoretically applied salmon CT (35 of 74 cells tested). Few neurons responded to salmon CT with an increase in firing rate (N = 3). When CGRP was iontophoretically applied a pattern of response resembling that of salmon CT was observed. CGRP was predominantly inhibitory and excited those neurons whose firing rate was increased by salmon CT. Inhibition was also the predominant effect of human CT. However, no neurons were excited by human CT. The results clearly demonstrate that a subpopulation of neurons with membrane sensitivity to salmon CT, human CT, and CGRP are present in the rat forebrain. This finding suggests that modulation of neuronal activity may underlie the behavioral and biochemical effects of these peptides when administered centrally. Endogenous CGRP and CT-like peptides in rat brain may be capable of regulating these events as neurotransmitters or neuromodulators.     

Distinct hemodynamic and gastric effects of human CGRP I and II in man

The human calcitonin gene-related peptides I and II (or and β) (CGRP I and II) are encoded by two different genes, but they have 34 of the 37 amino acid residues in common. Human CGRP I more potently stimulated blood flow through the skin and carotid artery (p<0.01), and the heart rate (p<0.05), and plasma renin activity and aldosterone secretion than human CGRP II (p<0.02). Inhibition of pentagastrin-stimulated gastric acid output, on the other hand, was only obtained with CGRP II. The separate effects of human CGRP I and II on the cardiovascular and gastric systems are presumably mediated by different receptors or receptor pathways recognized by the two closely related neuropeptides.     

The utility of 2-hydroxypropyl-beta-cyclodextrin as a vehicle for the intracerebral and intrathecal administration of drugs.

The substituted glucopyranose ring structure 2-hydroxypropyl-beta-cyclodextrin (CDEX) increases the solubility of molecules by inclusion of the agent in the lipophilic interior of the ring. This property is of particular use for the administration of molecules by the intracerebral (ICV) or intrathecal (IT) routes. In concentrations up to 40% w/v (isotonic), this agent (10 microliters) effect upon nociceptive or motor function after IT injection or on EEG and general behavior after ICV injection in rats. Using 20% CDEX, there is no change in the ED50 as compared to saline on the hot plate (HP) after IT injection of morphine, D-Ala2-D-Leu5 enkephalin or Tyr-Aib-Gly-gPhe-mAib-NH2, (Aib: alpha-aminoisobutyric acid) although there is an increase in their respective durations of effect. Cyclic peptide opioids: Tyr-c[D-A2bu-Gly-D-beta Nal(1)-D-Leu] (A2bu: alpha, gamma-diaminobutyric acid; beta-Nal(1): beta-naphthylalanine(1)) or Tyr-c[DA2bu-Gly-beta Nal(1)-D-Leu] are insoluble in saline but are readily dissolved in CDEX, and display a naloxone-sensitive antinociception following spinal administration. In other studies, saline insoluble capsaicin is administered in 25% dimethylsulfoxide (DMSO) or 20% CDEX (15 microliters; 5 mg/ml) which result in a significant reduction in the spinal levels of substance P and calcitonin gene related peptide and an increase in the HP latency. DMSO alone, but not CDEX alone, reduces the levels of the two peptides. These data emphasize the utility of complexation with CDEX for intracerebral drug delivery and compatibility with brain and spinal tissue.