Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

Cyclic GMP Regulates Nicotine-Induced Secretion from Cultured Bovine Adrenal Chromaffin Cells: Effects of 8–Bromo–Cyclic GMP, Atrial Natriuretic Peptide, and Nitroprusside (Nitric Oxide)

Methacholine, atrial natriuretic peptide (ANP), nitroprusside (nitric oxide), angiotensin II, and bradykinin raised cyclic GMP (cGMP) levels in cultured bovine adrenal chromaffin cells. The role of cGMP in secretion from chromaffin cells was examined using 8-bromo-cGMP. This analogue had no effect on basal secretion or secretion due to angiotensin II, bradykinin, or a high K+ level but potentiated secretion due to low doses of nicotine. At supramaximal doses of nicotine, 8-bromo-cGMP inhibited secretion. These effects of 8-bromo-cGMP were not due to changes in the nicotine-induced rise in cytosolic calcium concentration. A potentiation of secretion due to low doses of nicotine was also found following simultaneous addition of ANP or nitroprusside, a result suggesting that ANP and nitric oxide (endothelium-derived relaxing factor) could be important regulators of secretion from adrenal chromaffin cells.     

Elevation by Atrial Natriuretic Factors of Cyclic GMP Levels in Astroglia-Rich Cultures from Murine Brain

Atrial natriuretic factors, peptide hormones originally found in the heart, slowly but strongly elevate the level of cyclic GMP in primary astrocyte-rich cultures derived from brains of newborn rats or mice but not in neuron-rich cultures prepared from embryonic rat brain. In the absence of a phosphodiesterase inhibitor, a plateau level of cyclic GMP is obtained within 10 min. In the presence of the inhibitor 3-isobutyl-1-methylxanthine, the concentration of cyclic GMP continues to rise, even after 30 min. The elevation of the level of cyclic GMP in response to atrial natriuretic factor is much more pronounced in the rat cultures than the mouse cultures. Even at peptide concentrations of 1 microM, plateaus of the concentration-response curves are not yet reached. The potencies of the active peptides vary over a range of approximately 1.5 orders of magnitude, with atriopeptins II and III and auriculin A being the most potent ones. These results suggest (a) that atrial natriuretic factors may regulate functions of glial cells, most likely of astrocytes, in brain and (b) that such cultures may be useful tools in defining such astroglial functions.     

Atrial Natriuretic Hormones Raise the Level of Cyclic GMP in Neural Cell Lines

Atriopeptin III and related atrial natriuretic peptide hormones strongly elevate the level of cyclic GMP in three neural tumor cell lines. At peptide concentrations of 1 microM clear-cut plateaus of the dose-response curves are not yet reached. Atriopeptin III increases the intracellular concentration of cyclic GMP to a maximum in the course of 30-40 min. The effect of atriopeptin III on the cellular cyclic GMP level is independent of the concentration of extracellular Ca2+ and is not affected by the Ca2+ ionophore A23187. These results suggest (1) that atrial natriuretic hormones may play an important role in the nervous system, and (2) that cultured neural cells may be useful tools in the elucidation of the mechanisms of action of these hormones.     

C型钠尿肽及其受体在急性肺损伤大鼠肺组织的表达变化及其意义

目的了解C型钠尿肽及其受体NPRB在急性肺损伤大鼠肺组织中的表达变化规律。方法采用LPS注射建立ALI大鼠动物模型。将动物分为生理盐水组(N组),LPS干预1 h组(LPS 1 h组),LPS干预3 h组(LPS 3 h组),LPS干预6 h组(LPS 6 h组),通过RT-PCR检测各组大鼠肺组织CNP及NPRB mRNA的表达情况,以及免疫组化检测各组大鼠NPRB的表达变化,以生理盐水组作为阴性对照。结果正常大鼠肺组织可表达CNP及NPRB,LPS干预后,CNP显著升高,LPS 6 h达到高峰,与对照组比较有显著性差异(P0.05);相反,NPRB在LPS干预后出现表达降低,与对照组比较有显著性差异(P0.05)。结论CNP与NPRB的表达变化可能是导致肺损伤加重的重要原因之一。     

Atrial Natriuretic Factors Stimulate Accumulation and Efflux of Cyclic GMP in C6–2B Rat Glioma and PC12 Rat Pheochromocytoma Cell Cultures

Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6-2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates particulate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6-2B and PC12 cells with the rank order of potency being atriopeptin III = atriopeptin II greater than human atrial natriuretic polypeptide greater than atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6-2B cells fourfold and in PC12 cells 12-fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6-2B cells (47-fold) and PC12 cells (12-fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (250 microM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Natriuretic peptides in cardiovascular diseases

The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.     

Vasoactive Intestinal Peptide and Forskolin Stimulate Interleukin 6 Production by Rat Cortical Astrocytes in Culture via a Cyclic AMP-Dependent, Prostaglandin-Independent Mechanism

Abstract: In this study we analyzed the involvement of the cyclic AMP (cAMP)-protein kinase A system in the regulation of interleukin 6 production by cultured cortical astrocytes. Vasoactive intestinal peptide strongly increased, in a dose-dependent manner, interleukin 6 production. This effect was reduced when protein kinase A was blocked by KT-5720; it was not affected by calphostin C, a protein kinase C inhibitor. Forskolin caused a concentration-dependent increase in interleukin 6 release that was also inhibited by KT-5720. Because prostaglandins are believed to play a role in interleukin 6 production, we tried to determine whether the stimulatory effects of vasoactive intestinal peptide and forskolin on cytokine release might be mediated by stimulation of prostaglandin production in cortical astrocytes. Vasoactive intestinal peptide did not increase the production of either prostaglandin E₂ or F_2α. Conversely, forskolin concentration-dependently stimulated the production of both prostaglandins, an effect that was blocked by indomethacin. Indomethacin did not affect either vasoactive intestinal peptide- or forskolin-stimulated interleukin 6 production. To exclude the possibility that prostaglandins participate in interleukin 6 production induced by forskolin, we tested prostaglandins E₂ and F_2α. The former was completely ineffective in eliciting the cytokine production, whereas prostaglandin F_2α slightly increased interleukin 6 production only at the highest concentrations. 8-Bromo-cAMP and dibutyryl-cAMP stimulated interleukin 6 production to a lesser extent than vasoactive intestinal peptide and forskolin. In conclusion, we provide evidence that vasoactive intestinal peptide increases interleukin 6 production by astrocytes through the stimulation of the cAMP-protein kinase A pathway, an effect that is reproduced by cAMP analogues. In addition, we point out that prostaglandins are not involved in vasoactive intestinal peptide- and forskolin-mediated induction of interleukin 6 production in cultured astrocytes.     

Endothelin-3 reduces C-type natriuretic peptide-induced cyclic GMP formation in C6 glioma cells

The effect of endothelin-3 (ET-3) on C-type natriuretic peptide (CNP)-induced guanosine 3′,5′-cyclic monophosphate (cGMP) was examined in C6 glioma cells, CNP-induced cGMP formation was both time- and dose-dependent, with an EC₅₀ value of about 10 nM. While ET-3 and phorbol 12-myristate 13-acetate (PMA) had no effect on basal cGMP production, both compounds were potent inhibitors of CNP-induced cGMP formation, with IC₅₀ values of approximately 10 and 2 nM, respectively. Although protein kinase C (PKC) inhibitors had no effect on basal cGMP formation, Ro 31-8220, a PKC inhibitor, reversed the ET-3 inhibition on CNP-induced cGMP formation by 63% and that of PMA almost completely. Our findings suggest that stimulation of cGMP formation by CNP in C6 glioma cells is negatively modulated by PKC activation, and that the inhibitory action of ET-3 on CNP-stimulated cGMP formation is mediated partly by PKC.     

Noradrenaline Antagonizes and Ouabain Potentiates the Effects of iV-Methyl-D-Aspartate on Rat Cerebellar Cyclic GMP Production

Noradrenaline potently antagonizes the effects of N-methyl-D-aspartate (NMDA) (80 microM) on cyclic GMP production in immature rat cerebellar slices in vitro (IC50 = 0.6 microM). The effect is stereospecific (D-noradrenaline, IC50 = 100 microM), and also observed with adrenaline (IC50 = 0.5 microM) and isoprenaline (IC50 = 1.2 microM). The alpha 1-adrenoceptor agonists methoxamine or phenylephrine or the mixed alpha 1/alpha 2 agonists oxymetazoline or xylometazoline (100 microM) do not block the effects of NMDA, but the alpha 2-adrenoceptor agonist clonidine is weakly active (IC50 = 200 microM). Salbutamol and terbutaline were also inactive except at high concentrations (300 microM), as were a number of other catechol and phenylethylamine derivatives. The antagonistic effects of noradrenaline on the NMDA response were insensitive to phentolamine, atenolol, or propranolol (up to 100 microM), but were blocked by the alpha 2 antagonist idazoxan (1-10 microM). The Na+,K+-ATPase inhibitor ouabain (0.1-10 microM) markedly potentiates the effects of NMDA in this model, and also antagonizes and reverses the ability of noradrenaline (10 microM) to block the effects of NMDA. The results suggest that noradrenaline and Na+,K+-ATPase activity have potent modulatory effects on the NMDA response.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.     

Angiotensin-Induced Cyclic GMP Production Is Mediated by Multiple Receptor Subtypes and Nitric Oxide in N1E-115 Neuroblastoma Cells

Angiotensin II (AngII) elicited a rapid and dose-related production of intracellular cyclic GMP (cGMP) in murine neuroblastoma N1E-115 cells. The agonist-induced rise in cGMP levels was blocked in a monophasic fashion by the AT1-selective antagonist DuP 753 or the nonselective antagonist [Sarc1,Ile8]-AngII, and both antagonists produced complete inhibition of the cGMP response elicited by submaximal concentrations of AngII. In contrast, the AT2-selective antagonist CGP 42112A inhibited the cGMP response biphasically. At lower antagonist concentrations, agonist-induced cGMP production was only partially inhibited, whereas complete inhibition was observed only when the concentration of CGP 42112A was increased sufficiently to interact with both AT1 and AT2 receptor subtypes. AngII also increased inositol trisphosphate (InsP3) levels in N1E-115 cells. However, the InsP3 response was mediated exclusively by the AT1 receptor subtype because it was inhibited by lower, AT1-selective concentrations of DuP 753, whereas only higher, nonselective concentrations of CGP 42112A were effective. Finally, the stimulatory effects of AngII on cGMP production appeared to be mediated by the intracellular formation of nitric oxide in that they were attenuated by the nitric oxide synthase inhibitor, N-monomethyl-L-arginine. Collectively, these results suggest that the AngII-elicited rise in cGMP levels may require an interaction between AT1-mediated mobilization of intracellular Ca2+, as well as some partial role of AT2 receptors.     

Regulation of Muscarinic Receptor-Mediated Cyclic GMP Synthesis by Cultured Mouse Neuroblastoma Cells

Mouse neuroblastoma clone N1E-115 has muscarinic acetylcholine receptors that mediate cyclic GMP synthesis. This receptor-mediated response is not significantly higher than background until the cells have been maintained in the stationary phase for at least 1 week. The basis of the influence of time in culture on the cyclic GMP response was investigated. The relative amount of cyclic GMP synthesized by intact cells was measured by radioactively labeling the GTP pool with [³H]guanine, incubating cells with agonists, and then chromatographically isolating [³H]cyclic GMP. Carbamylcholine-, ionophore X-537A-, and sodium azide-induced cyclic GMP formation increased with time in culture to a maximum of 13-, 9-, and 2.5-fold above basal, respectively. There was no change in the number or the apparent affinity of the muscarinic receptors as measured by [³H]quinuclidinyl benzylate ([³H]QNB) binding. In addition, there was no change in the apparent affinity of the receptors for agonist as measured by the ability of carbamylcholine to displace the specific binding of [³H]QNB. Guanylate cyclase activity per milligram protein and per cell in-creased six- and sevenfold, respectively, from day 0 to day 22. However, this increase in guanylate cyclase appeared to precede the marked increase in sensitivity of the cells to agonists. These data suggest that, in addition to guanylate cyclase and muscarinic receptors, there is another factor which is responsible for the development of this muscarinic receptor-mediated response.     

Cyclic GMP Inhibits a Pharmacologically Distinct Na+/H+ Exchanger Variant in Cultured Rat Astrocytes via an Extracellular Site of Action

Abstract: There is growing evidence that cyclic GMP (cGMP) plays important roles in the brain. In cultured rat astrocytes, we observed that the cGMP-inducing C-type natriuretic peptide (CNP) and cGMP analogues caused a decrease in intracellular pH (pH_i). To examine whether this effect was due to inhibition of an Na⁺/H⁺ exchanger (NHE), we acidified cells by replacing extracellular Na⁺ by choline and examined the kinetics of the pH_i recovery that occurred on reintroduction of Na⁺ in the extracellular medium. Both CNP and amiloride analogues inhibited the Na⁺-dependent pH_i recovery, even in the nominal absence of CO₂/HCO₃^?. This indicated that CNP inhibited the activity of an exchanger that was Na⁺-dependent, HCO₃^?-independent, and sensitive to known inhibitors of NHE. However, comparison of the potencies of four distinct amiloride analogues revealed a pharmacological profile that was different from that of any other NHE characterized to date. cGMP mimicked the effect of CNP on sodium-dependent pH_i recovery, but the native nucleotide was as potent as membrane-permeant analogues. Intracellularly produced cGMP was very rapidly exported out of astrocytes. Probenecid and niflumic acid slowed down the rate of cGMP egression and inhibited the effect of CNP on Na⁺-dependent recovery, but not that of extracellular cGMP. Altogether, our data indicate that cGMP inhibits a novel type of NHE in astrocytes via an extracellular site of action. If these results with primary cultures transfer to brain, this phenomenon may constitute a mechanism by which natriuretic peptides exert some of their actions in the brain, as pH_i transients have been shown to modulate several important astrocytic functions.     

Genetic Expression of Cyclic GMP Phosphodiesterase Activity Defines Abnormal Photoreceptor Differentiation in Neurological Mutants of Inherited Retinal Degeneration

We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death.     

Relationship Between Cycles in Level of Serotonin N-Acetyltransferase Activity and Cyclic GMP Content of Cultured Chick Pineal Glands

We examined effects of supplements on cycles in cyclic GMP content and serotonin N-acetyltransferase (NAT) activity in cultured chick pineal glands. Increases in cyclic GMP content and NAT activity were stimulated by 1-ethyl-4(isopropylidene-hydrazino)-1H-pyrazolo[3,4-b]pyridene-5-c arboxylic acid, ethyl ester, hydrochloride and isobutylmethyl xanthine under diurnal illumination and in constant darkness, but subsequent decreases were not inhibited. Hypoxanthine had little effect on NAT activity under all lighting conditions, or on the content of cyclic GMP in glands cultured in the dark. However, it markedly stimulated accumulation of cyclic GMP in illuminated cultures. EGTA or additional Ca2+ had no effect on pineal NAT activity. However, EGTA markedly stimulated accumulation of cyclic GMP both in the light and in the dark. Supplementary Ca2+ slightly retarded accumulation of cyclic GMP in the dark but stimulated slightly in the light.     

Dexamethasone Up-Regulates a Constitutive Nitric Oxide Synthase in Cerebellar Astrocytes but Not in Granule Cells in Culture

Abstract: Treatment of rat cerebellar astrocyte-enriched primary cultures with dexamethasone enhances the nitric oxide-dependent cyclic GMP formation induced by noradrenaline in a time-(>6 h) and concentration-dependent manner (half-maximal effect at 1 n M ). Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [³H]arginine to [³H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up-regulate astroglial calcium-dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase and are the first report of a differential long-term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities.     

Astrocytes from Forebrain, Cerebellum, and Spinal Cord Differ in Their Responses to Vasoactive Intestinal Peptide

Astrocytes from cortex, cerebellum, and spinal cord responded to isoproterenol and vasoactive intestinal peptide (VIP) with increases in intracellular cyclic AMP levels. The response to VIP was as great as that to isoproterenol in cortical astrocytes (180-fold and 185-fold, respectively), and the effect of VIP in combination with isoproterenol was partially additive. Spinal cord astrocytes also responded to VIP and isoproterenol with equal potency (seven- to ninefold and eight- to 13-fold, respectively), but the level of response was much smaller than in cortex. Spinal cord astrocytes were synergistic in their response to VIP and isoproterenol. The response to VIP was lowest in cerebellar astrocytes (only threefold), and no additivity was observed when VIP was added together with isoproterenol. A small response to alpha-melanocyte stimulating hormone (alpha-MSH) was also observed in cortex and cerebellum, but not in spinal cord. Somatostatin inhibited the response to isoproterenol in cortex and cerebellum, but had no effect in spinal cord. The results from the above study show that astrocytes obtained from these three regions of the rat CNS express quite different responses to VIP and alpha-MSH and further point to possible astrocyte heterogeneity.