Production of cAMP by adrenomedullin in human oligodendroglial cell line KG1C: comparison with calcitonin gene-related peptide and amylin.

The actions and the presence of adrenomedullin (AM) were investigated in cultured human oligodendroglial cell line KG1C. AM and AM mRNA were detected in KG1C cells by immunohistochemistry and RT-PCR. mRNAs for calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs) 1, 2 and 3 but not for calcitonin receptors were detected in the cells, while mRNAs for CRLR, calcitonin receptors and all RAMPs were detected in the human cerebellum. Application of AM resulted in time- and concentration-dependent increases in the cAMP level of KG1C cells. Calcitonin gene-related peptide (CGRP) and amylin, peptides structurally related to AM, also increased cAMP. The potencies for the cAMP production of the three peptides were CGRP > or =AM > amylin with EC(50) of 8, 18, 90 nM, respectively. The responses induced by AM were strongly inhibited by the CGRP(1) receptor antagonist human CGRP(8-37), and inhibited also by the AM receptor antagonist human AM(22-52). In contrast, the responses induced by CGRP or amylin were inhibited only by CGRP(8-37) and not by AM(22-52). The responses induced by all three peptides were unaffected by the amylin receptor antagonist human amylin(8-37). The CGRP(2) receptor agonist human [Cys(Acm)(2,7)]CGRP significantly increased the cAMP level but the increase was smaller than that caused by CGRP. This increase in cAMP was unaffected by CGRP(8-37), AM(22-52) or by amylin(8-37). These results suggest that in KG1C cells, AM increases cAMP through AM and CGRP(1) receptors, whereas CGRP does so through CGRP(1) and CGRP(2) receptors, and amylin exerts its effects through CGRP(1) receptors. Collectively, these findings imply that AM released from oligodendroglial cells may play a role in the regulation of oligodendrocytes via autocrine/paracrine through AM receptors and CGRP(1) receptors.     

Adrenomedullin-2/Intermedin Induces cAMP Accumulation in Dissociated Rat Spinal Cord Cells: Evidence for the Existence of a Distinct Class of Sites of Action

Adrenomedullin-2/intermedin is structurally related to the calcitonin family of peptides, which includes calcitonin gene-related peptide (CGRP), adrenomedullin, and amylin. We recently reported that CGRP and adrenomedullin act through distinct receptors to induce cyclic adenosine monophosphate (cAMP) accumulation in dispersed cells from embryonic rat spinal cord. Here, we investigated the apparent affinity and efficacy of adrenomedullin-2/intermedin for these receptors. Adrenomedullin-2/intermedin competed with [(125)I]-CGRP for binding to specific embryonic spinal cord cells with a pIC(50) of 9.73 +/- 0.06. Interestingly, adrenomedullin-2/intermedin competed for specific [(125)I]-adrenomedullin binding in a biphasic manner with pIC(50) of 9.03 +/- 0.22 and 6.45 +/- 0.24, respectively. Cellular levels of cAMP were increased by adrenomedullin-2/intermedin (pEC(50) 7.84 +/- 0.08) when cells were exposed to this peptide for 10 min at 37 degrees C. This effect was partially inhibited by the non-peptide antagonist BIBN4096BS (pA(2) 6.56 +/- 0.12), the adrenomedullin antagonist hAM(22-52) (pA(2) 6.36 +/- 0.30), and the adrenomedullin/CGRP antagonist CGRP(8-37) (pA(2) 7.24 +/- 0.60). More interestingly, a highly significant effect of adrenomedullin-2/intermedin on cAMP accumulation (pEC(50) 7.3 +/- 0.14) was still observed even in the presence of a mixture of saturating concentrations of BIBN4096BS, hAM(22-52), and the amylin antagonist AC187. Taken together, these data provide evidence for the possible existence of a distinct class of receptor sites for adrenomedullin-2/intermedin in embryonic rat spinal cord cells.     

Adrenomedullin,a novel vasoactive hormone,binds to mouse astrocytes and stimulates cyclic AMP production

We have examined the effects of adrenomedullin (AM), a novel hypotensive peptide first isolated from human pheochromocytoma, on receptor binding and cyclic AMP (cAMP) generation in primary cultures of mouse astrocytes. Competition binding studies showed that rat adrenomedullin (rAM) displaced the specific binding of [¹²⁵I]rAM in a dose-dependent manner, with an estimated IC₅₀ of 33 nM. Rat calcitonin gene-related peptide (rCGRP), which interacts with AM receptors in some vascular tissues, did not produce significant displacement of [¹²⁵I]rAM at concentrations up to 3.3 μM. rAM stimulated cAMP production in mouse astrocytes in a dose-dependent manner, with an EC₅₀ of 74 nM and a maximal stimulatory concentration of 1 μM. CGRP_8–37, a CGRP receptor antagonist, failed to inhibit the cAMP response to rAM, although it attenuated CGRP-stimulated cAMP production. These data indicate that cultured mouse astrocytes possess specific AM receptors which are coupled to adenylate cyclase but do not interact with CGRP. AM may function as a neuropeptide and may play a role in the central regulation of blood pressure and body fluid balance. © 1996 Wiley-Liss, Inc.     

Functional calcitonin gene-related peptide type 1 and adrenomedullin receptors in human trigeminal ganglia, brain vessels, and cerebromicrovascular or astroglial cells in culture.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent dilators of human brain arteries, and they have been implicated in the neurogenic inflammation underlying migraine headache and in the evolution of stroke, respectively. However, little is known about the presynaptic and postsynaptic distribution of their respective receptors in the human cerebrovascular bed and trigeminovascular system. In the current study, the expression of mRNA for ADM and the two cloned human CGRP1 receptors (identified here as A-CGRP1 receptors [Aiyar et al., 1996] and K-CGRP1 receptors) [Kapas and Clark, 1995] were evaluated in human brain vessels and trigeminal ganglia. Further, the ability of CGRP and ADM to activate adenylate cyclase in cerebromicrovascular and astroglial cell cultures was determined, and the receptors involved were characterized pharmacologically. Isolated human pial vessels, intracortical microvessels, and capillaries, as well as cultures of brain endothelial (EC), smooth muscle (SMC), and astroglial (AST) cells, all expressed mRNA for the two cloned CGRP1 receptors; however, message for the K-CGRP1 receptor was barely detectable in microvascular tissues and cells. In contrast, only isolated capillaries and cultured AST exhibited message for the ADM receptor. In human trigeminal ganglia, mRNA for ADM and the two CGRP1 receptors was systematically present. The CGRP dose-dependently increased (up to 50-fold) cAMP formation in cell cultures, an effect significantly blocked by 0.1 to 10 micromol/L of the CGRP1 receptor antagonist CGRP8-37. The ADM receptor agonist, ADM13-52 (1 micromol/L), similarly increased cAMP production in all cell types, and this response was virtually abolished by 1 micromol/L CGRP8-37. Low concentrations (1 to 10 micromol/L) of the ADM receptor antagonist ADM22-52 blocked the ADM13-52-induced cAMP formation in AST (26% at 10 micromol/L, P < 0.05), whereas they potentiated this response in brain EC and SMC (40% and 100%, P < 0.001, respectively). Even at a higher dose (50 micromol/L), ADM22-52 inhibited the ADM13-52 effect in vascular cells (45%) much less effectively than in AST (95%). These results indicate that both CGRP and ADM can affect human brain vessels through a CGRP1 receptor, and they further suggest the presence of functional ADM receptors in human astroglial cells.     

Bradykinin receptors of cerebral microvessels stimulate phosphoinositide turnover.

We examined by ligand binding methods whether bradykinin (BK) receptors exist in rat and pig cerebral microvessels, and in the cerebral cortex from which the microvessels were isolated. We found a high-affinity and saturable BK receptor site in both rat and pig cerebral microvessels, but not in their cerebral cortex. The maximal density of binding and the dissociation constant were 8.0 +/- 4.1 and 6.8 +/- 1.5 fmol/mg of protein and 47 +/- 24 and 150 +/- 8 pM (mean +/- SD) in cerebral microvessels of the pig and rat, respectively. The high-affinity specific binding of BK was effectively displaced by des-Arg0[Hyp3-Thi5-8,D-Phe7]BK, a specific B2 receptor antagonist, but not by des-Arg9[Leu8]BK, a specific B1 antagonist. We also demonstrated that BK increases phosphatidylinositol hydrolysis in cerebral microvessels of the rat and pig. This effect was also blocked by the B2, but not by the B1, antagonist. Increased phosphatidylinositol hydrolysis was manifested by a rapid transient increase in inositol trisphosphate and the later slow accumulation of inositol bisphosphate and inositol monophosphate. Preincubation of microvessels with phorbol ester, stable GTP analogs, pertussis toxin, or in Ca(2+)-free buffer did not influence BK activation of phosphatidylinositol hydrolysis. These results demonstrate the existence of BK receptors of the B2 subtype in brain microvessels, which may play an important role in modulation of the brain microcirculation, probably via increased phosphoinositide turnover.     

Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastboma × glioma hybrid cells (NG108-15)

Adrenomedullin (ADM) is a hypotensive peptide with structural homology, including a ring structure linked by a disulfide bridge, to calcitonin gene-related peptide (CGRP), calcitonin and amylin. ADM is predominantly synthesized in the adrenal medulla, but immunoreactive ADM has also been detected in the human brain. Here we have characterized ADM binding sites in cultured rat astrocytes using human [¹²⁵I]ADM(1–52) as radioligand. Half-maximal inhibition of [¹²⁵I]ADM(1–52) binding by intact rat ADM(1–50) amounted to 0.27 ± 0.03 nM (n = 15). The related peptides rat α-CGRP, rat amylin and salmon calcitonin displaced [¹²⁵I]ADM(1–52) at 85-, 148-, and > 4000-fold higher concentrations. Half-maximal stimulation of CAMP accumulation by rat ADM(1–50) was obtained with 1.00 ± 0.12 nM (n = 16). Rat α-CGRP was 214-fold, and rat amylin and salmon calcitonin were > 1000-fold less potent. Concerning cAMP accumulation the results were indistinguishable in mouse neuroblastoma × rat glioma hybrid cells (NG108-15), but here rat α-CGRP was > 1000-fold less potent than rat ADM(1–50). Human ADM(22–52) and human CGRP-I(8–37), which lack the ring structure, failed to stimulate cAMP accumulation, but they antagonized rat ADM(1–50) stimulated cAMP accumulation with inhibitory constants of 365 ± 93 nM and 92 ± 2 nM in astrocytes, and 45 ± 3 nM and 1300 ± 500 nM in NG108-15 cells. Rat ADM(1–50) did not raise cytosolic free calcium concentrations in astrocytes and NG108-15 cells. In conclusion, we have identified novel ADM receptors coupled to cAMP formation in cultured rat astrocytes and NG108-15 cells. Different interactions with the homologous peptide CGRP as well as the truncated receptor antagonists ADM(22–52) and CGRP(8–37) in rat astrocytes and neuroblastoma × glioma hybrid cells are consistent with ADM receptor isotypes in the brain.     

Sustained, long-lasting inhibition of nitric oxide synthase aggravates the neural damage in some models of excitotoxic brain injury

To determine pharmacological specificity of intracerebroventricular (IVT) administration of adrenomedullin (AM) on water and sodium excretion, studies were performed in rats pretreated with AM (22-52), a putative AM-receptor antagonist or CGRP(8-37), a ligand that preferentially antagonizes the CGRP(1)-receptor subtype. In addition, the effects of IVT injection of calcitonin-gene-related peptide (CGRP) on urinary water and electrolyte excretion was assessed. Intracerebroventricular administration of rat adrenomedullin to conscious hydrated rats resulted in a significant increase in urinary volume and sodium excretion during the 6-h period of urine collection and was most effective at 3 and 6 h. Although less effective than AM, central administration of CGRP induced diuresis and natriuresis. Pretreatment with AM (22-52) or CGRP(8-37) significantly suppressed the diuretic and natriuretic effect of IVT-AM. These data suggest that both CGRP(1) and AM receptors are involved in the centrally mediated diuretic and natriuretic action of the AM. Our results provide evidence supporting the hypothesis that endogenous AM plays a role in the central nervous control of fluid and electrolyte homeostasis.     

Bace 1

Adrenomedullin (AM) has two specific receptors formed by the calcitonin-receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3. These are known as AM₁ and AM₂ receptors, respectively. In addition, AM has appreciable affinity for the CGRP₁ receptor, composed of CL and RAMP1. The AM₁ receptor has a high degree of selectivity for AM over CGRP and other peptides, and AM_22–52 is an effective antagonist at this receptor. By contrast, the AM₂ receptor shows less specificity for AM, having appreciable affinity for βCGRP. Here, CGRP_8–37 is either equipotent or more effective as an antagonist than AM_22–52, depending on the species from which the receptor components are derived. Thus, under the appropriate circumstances it seems that βCGRP might be able to activate both CGRP₁ and AM₂ receptors and AM could activate both AM₁ and AM₂ receptors as well as CGRP₁ receptors. Current peptide antagonists are not sufficiently selective to discriminate between these three receptors. The CGRP-selectivity of RAMP1 and RAMP3 may be conferred by a putative disulfide bond from the N-terminus to the middle of the extracellular domain of these molecules. This is not present in RAMP2.     

Blockade of adrenomedullin receptors reverses morphine tolerance and its neurochemical mechanisms

Adrenomedullin (AM) has been demonstrated to be involved in the development of opioid tolerance. The present study further investigated the role of AM in the maintenance of morphine tolerance, morphine-associated hyperalgesia and its cellular mechanisms. Intrathecal (i.t.) injection of morphine for 6 days induced a decline of its analgesic effect and hyperalgesia. Acute administration of the AM receptor antagonist AM_22-52 resumed the potency of morphine in a dose-dependent manner (12, 35.8 and 71.5 μg, i.t.). The AM_22-52 treatment also suppressed morphine tolerance-associated hyperalgesia. Furthermore, i.t. administration of AM_22-52 at a dose of 35.8 μg reversed the morphine induced-enhancement of nNOS (neuronal nitric oxide synthase) and CGRP immunoreactivity in the spinal dorsal horn and/or dorsal root ganglia (DRG). Interestingly, chronic administration of morphine reduced the expression of the endogenous opioid peptide bovine adrenal medulla 22 (BAM22) in small- and medium-sized neurons in DRG and this reduction was partially reversed by the administration of AM_22-52 (35.8 μg). These results suggest that the activation of AM receptors was involved in the maintenance of morphine tolerance mediating by not only upregulation of the pronociceptive mediators, nNOS and CGRP but also the down-regulation of pain-inhibiting molecule BAM22. Our data support the hypothesis that the level of both pronociceptive mediators and endogenous pain-inhibiting molecules has an impact on the potency of morphine analgesia. Targeting AM receptors is a promising approach to maintain the potency of morphine analgesia during chronic use of this drug.     

Expression of spinal cord Fos protein in response to intrathecal adrenomedullin and CGRP in conscious rats

Adrenomedullin (AM) immunoreactivity and mRNA, in addition to a large number of specific AM-binding sites, exist in the rat spinal cord. However, no phenotype has been reported for AM in the spinal cord. Here, expression of c-fos in response to intrathecal (i.t.) administration of AM, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) was examined in the thoracic, lumbar and sacral regions of spinal cord in conscious rats. Two hours after i.t. administration of either CGRP (2.5 and 10 microg) or AM (10 microg), the number of c-Fos immunoreactive nuclei was increased in all the spinal regions examined in this study, with the highest increase observed in the superficial dorsal horn. Few cells with c-fos immunoreactivity were found in the spinal cord of rats 2 h after i.t. injection of either saline or PAMP. Effects of AM (10 microg) and CGRP (2.5 microg) on c-fos expression were blocked when rats were pretreated with 40 microg of intrathecal CGRP8-37 (CGRP1 receptor antagonist). Fos-like immunoreactivity induced by i.t. CGRP and/or AM were also significantly abolished by i.t. administration of the nitric oxide (NO) inhibitor, l-NAME, indicating that endogenous NO is a necessary intermediary in CGRP and AM induced c-fos expression in the rat spinal cord. In conclusion, AM induces c-fos expression in rat spinal cord when administered intrathecally, with the pattern being similar to those produced by i.t. CGRP. Effects of the two peptides are sensitive to CGRP8-37 and l-NAME.     

Pre-treatment of adrenomedullin suppresses cerebral edema caused by transient focal cerebral ischemia in rats detected by magnetic resonance imaging

Recent studies suggest the protective effects of adrenomedullin (AM) on ischemic brain damage. The present study was aimed at investigating the effects of AM and its receptor antagonist, AM_22-52, on ischemia-induced cerebral edema and brain swelling in rats using magnetic resonance imaging. Rats were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. Intravenous injection of AM (1.0 μg/kg), AM_22-52 (1.0 μg/kg), or saline was made before MCAO. Effects of AM injection just after reperfusion were also investigated. One day after ischemia, increases in T₂-weighted signals in the brain were clearly observed. Total edema volume, as well as brain swelling, was greatly and significantly reduced by pre-treatment of AM (reduced by 53%). Extent of brain swelling was significantly correlated with the volume of cerebral edema. The protective effect of AM against edema was more clearly observed in the cerebral cortex (reduced by 63%) than the striatum (reduced by 31%). Increased T₂ relaxation time in the cortex was recovered partially by pre-treatment of AM. Post-treatment of AM had no effects. Pre-treatment of AM_22-52 tended to exacerbate the edema. In another line of experiment, cocktail administration of AM with melatonin, a pineal product having neuroprotective potential as a free radical scavenger, failed to enhance the protective effects of AM alone. The present study clearly suggests the prophylactic effects of AM against cerebral edema, especially the cortical edema, in a rat stroke model.     

A biochemical and morphological study of the altered growth pattern of central catecholamine neurons following 6-hydroxydopamine

The ability of a series of adrenergic agents to displace the binding to brain membranes of [3H]WB 4101, a potent alpha-adrenergic antagonist (WB 4101 = 2-[2-(2,6-dimethoxyphenoxy)ethylaminomethyl]-1,4-benzodioxane hydrochloride), has been compared with the potency of these agents in stimulating or inhibiting the alpha-adrenergic component of cyclic AMP accumulation in rat cerebral cortex slices. [3H]WB 4101 rapidly bound to a high affinity site (KD = 2.7 nM) in membranes from cerebral cortex. Binding came to equilibrium by 2 min at 37 degrees C and was rapidly reversed in the presence of phentolamine. The potencies of adrenergic agents (WB 4101 greater than phentolamine greater than naphazoline) in displacing binding of [3H]WB 4101 were comparable to the potencies of these agents as inhibitors of the alpha-adrenergic component of norepinephrine-stimulated cyclic AMP accumulations. Phenoxybenzamine, clonidine, chlorpromazine and haloperidol were about 10--30 times more potent in inhibiting cyclic AMP accumulation than in displacing [3H]WB 4101 binding. The potency of classical alpha-adrenergic agonists in displacing WB 4101 (epinephrine greater than norepinephrine greater than methoxamine) correlated with the ability of these agonists to increase cyclic AMP levels. Overall a significant correlation (r = 0.87, P less than 0.005) was found between WB 4101 binding and alpha-adrenergically mediated cyclic AMP accumulation in brain. Several ligands bind to specific sites in brain membranes with alpha-adrenergic receptor properties. The identification of these binding sites as receptors depends on a correlation of binding with a known alpha-adrenergic receptor-mediated response in brain. These data demonstrating that WB 4101 correlates with norepinephrine-stimulated cyclic AMP accumulation suggest that WB 4101 may bind to the membrane receptor sites mediating the alpha-adrenergic accumulation of cyclic AMP in rat cerebral cortex.     

Beta-adrenergic receptor activity of cerebral microvessels in experimental diabetes mellitus.

The effect of diabetes mellitus on beta-adrenergic receptor number (B(max)), receptor-cyclase coupling and adenylate cyclase (AC) activity was determined in cerebral microvessels isolated from control and streptozotocin induced diabetic rats after 5 weeks of induction of diabetes. Scatchard analysis of [125I]iodocyanopindolol (ICYP) binding indicated that the B(max) (fmol/mg) in diabetic rat cerebral microvessels (63.8 +/- 4.8) (mean +/- S.E.M.) was not significantly different from the B(max) in control rats (56.5 +/- 6.9). Isoproterenol competition of [125I]ICYP binding sites indicated that the percentage of beta-receptors expressing high affinity binding was 53.9 +/- 0.45% in control rats and 47.5 +/- 2.3% in diabetic rats. The total isoproterenol-stimulated AC activity (pmol cAMP/mg) in diabetic rats (76.7 +/- 6.1) was significantly lower than that in control rats (118.4 +/- 11.2) (P less than 0.01). However, the net isoproterenol-stimulated AC activity (i.e. total minus GTP-stimulated AC activity) was not altered in diabetes. The net sodium fluoride (NaF) stimulated AC activity in diabetic rats (109.5 +/- 11.4) was significantly lower than the control rats (154.3 +/- 16.3) (P less than 0.05). It is concluded that diabetes mellitus in rats is associated with reduced post receptor activation of adenylate cyclase in cerebral microvessels while the beta-adrenergic receptor density, affinity and receptor-cyclase coupling are not significantly altered.     

Effect of age on β-adrenergic receptors on cerebral microvessels

The responsiveness of β-adrenergic receptors in cerebral microvessels was studied in aged rats by measuring cAMP formation induced by norepinephrine and [¹²⁵I]iodohydroxybenzylpindolol binding. The density of β-receptor sites is reduced by aging in the rat cerebral microvessels. These results suggest that the changes in brain circulation during aging may be at least partially due to a reduction of the β-adrenergic control of cerebral microvessels.     

Calcitonin gene-related peptide rapidly downregulates nicotinic receptor function and slowly raises intracellular Ca2+ in rat chromaffin cells in vitro.

Although calcitonin gene-related peptide (CGRP) modulates muscle-type nicotinic acetylcholine receptors (nAChRs) via intracellular second messenger-mediated phosphorylation, the action of this peptide on neuronal-type nAChRs remains unknown. Using neuronal nAChRs of rat chromaffin cells in vitro we studied the effect of CGRP, which is physiologically present in adrenal medulla, on membrane currents and [Ca2+]i transients elicited by nicotine. Our main novel observation was that CGRP (either bath-applied or focally applied for a few seconds or even co-applied with nicotine for a few milliseconds) selectively and rapidly blocked nAChRs (a phenomenon unlikely caused by intracellular messengers in view of its speed) without affecting GABA receptors. The inhibitory effect of CGRP was independent of [Ca2+]i or membrane potential and not accompanied by baseline current changes. Like the competitive antagonist N,N,N-trimethyl-1-(4-trans-stilbenoxy)-2-propilammonium, CGRP induced a rightward, parallel shift of the nicotine dose-response curve; during co-application of these blockers the nicotine dose-ratio value was the sum of the values obtained with each antagonist alone. The block by CGRP was insensitive to the receptor antagonist hCGRP8-37 but mimicked by CGRP1-7. Persistent application of CGRP slowly increased [Ca2+]i, a phenomenon independent from external Ca2+, thus implying Ca2+ release from internal stores, and suppressed by hCGRP8-37. CGRP1-7 had no significant effect on [Ca2+]i. We propose that the 1-7 amino acid sequence of CGRP was responsible for the direct, rapid block of nAChRs, whereas the full-length peptide molecule was necessary for the delayed rise in internal Ca2+ potentially able to trigger phosphorylation-dependent modulation of nicotinic receptor function.     

Alpha-1 adrenergic receptor binding and adrenergic-stimulated cyclic AMP production in rat cerebral cortex after chronic lithium treatment

Summary Administration to rats of dietary lithium for 30 days was followed by evaluation of alpha-1 adrenergic receptor binding and of adrenergic-stimulated cyclic AMP (cAMP) accumulation in rat cerebral cortex. Chronic lithium treatment did not alter the binding characteristics of [³H]prazosin or the proportion of alpha-1 adrenergic receptor subtypes distinguished by chlorethylclonidine (CEC) pretreatment in rat cerebral cortical membranes. Accumulation of cAMP in cortical slices incubated with adrenergic agonists was unaffected in lithium-treated rats. These results demonstrate that chronic lithium treatment-induced reduction of norepinephrine (NE)-stimulated phosphoinositide (PI) hydrolysis (Casebolt and Jope, 1987) is not due to changes in the alpha-1 adrenergic receptor and is more sensitive to in vivo lithium treatment than is adrenergic-stimulated cAMP accumulation.     

Adenosine receptors of cerebral microvessels and choroid plexus

We studied, by ligand binding methods, the two adenosine receptors, A1 and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]L-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'-N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity (Kd approximately 250 nM) and low-affinity (Kd approximately 1-2 microM) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these micro-vessels.     

Neuropeptide effects on rat chondrocytes and perichondrial cells in vitro

This study examines if cultured chondrocytes and perichondrial cells change the level of cAMP and/or cGMP in response to application of the neuropeptide calcitonin gene-related peptide (CGRP). Cells collected from the knee region of 4-8 days old rat pups were cultured in vitro. Cultures were exposed to 10(-10)-10(-6) M CGRP during 10 minutes. The levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the cultures and in controls were determined by radioimmunoassay. The results show that application of CGRP causes a distinctly increased level of cAMP, that was absent when CGRP was applied together with the CGRP(1) receptor antagonist. The level of cGMP was not obviously altered. Hence, it is possible that terminals of primary sensory neurones present in developing cartilage influence chondrocytes and perichondrial cells via local release of CGRP.     

Roles of calcitonin gene-related peptide and its receptors in pain-related behavioral responses in the central nervous system

Calcitonin gene-related peptide (CGRP) is a 37 amino-acid peptide, which is widely distributed in peripheral and central nervous system. There are two types of CGRP receptors, CGRP receptor 1 and CGRP receptor 2. It is known that CGRP plays important roles in multiple physiological processes. Studies demonstrate that CGRP and CGRP receptors are involved in the transmission and modulation of pain information in peripheral and central nervous system. CGRP8-37, a specific antagonist for CGRP receptor 1, is widely used to differentiate the two typical CGRP receptors. There are two ambiguous points about the effects of CGRP and CGRP8-37 on pain-related behavioral responses. The first is the effects of exogenous CGRP and CGRP8-37 on the transmission and regulation of pain information in the spinal cord. The second is the effects of these peptides in pain modulation at super-spinal levels. The specific goal of this review is to summarize the roles of CGRP, CGRP8-37 and CGRP receptors in pain-related behavioral responses in the central nervous system.