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.     

Neuromedin S exerts an antidiuretic action in rats

We recently identified neuromedin S (NMS) as an endogenous ligand for the FM-4/TGR-1 receptor. Here, we examined the possible involvement of central NMS in regulation of urinary output and vasopressin (AVP) release in rats. Intracerebroventricular (icv) injection of NMS induced a dose-dependent increase in the plasma level of AVP, followed by a decrease of nocturnal urinary output. Expression of cFos after icv injection of NMS was observed in the supachiasmatic nucleus (SCN), arcuate nucleus, paraventricular nucleus (PVN), and supraoptic nucleus (SON). The cFos expressing cells in PVN and SON, but not SCN, were then double-stained using antibodies against the vasopressin. On the other hand, icv injection of neuromedin U, which also binds to the FM-4/TGR-1 receptor, required a concentration ten times higher than that of NMS in order to exert the same antidiuretic potency. These results suggest that central NMS may exert a physiological antidiuretic action via vasopressin release.     

In vivo effect of pituitary adenylate cyclase activating polypeptide 38 (PACAP 38) on the secretion of luteinizing hormone (LH) in male rats.

In view of the recent demonstrations that pituitary adenylate cyclase activating polypeptide (PACAP) 38 stimulates the release of LH from superfused pituitary cells and that the hypothalamus and anterior pituitary have highly selective binding sites for the peptide, we have surveyed the effect of intraatrial injections of PACAP 38 and vasoactive intestinal peptide (VIP), which has 68% homology with PACAP 38, in intact adult male rats. Furthermore the effect of intracerebroventricular (icv) injection of PACAP 38 was investigated. Intraatrial (10, 30, 100 micrograms) and icv (8, 32 micrograms) administration of PACAP 38 stimulated LH release significantly (P less than 0.01) in a dose-related fashion. Icv injection at a dose of 0.8 microgram was ineffective. The time course pattern of LH release by intraatrial injection and that by icv injection was similar, but the LH levels increased by intraatrial injection were much higher than that by icv injection. Intraatrial administration of VIP had almost no effect on LH release. These findings suggested that PACAP 38 stimulates LH release in vivo.     

Characterization of specific binding of atrial natriuretic peptide (ANP) to rat PC12 phaeochromocytoma cells

Specific binding sites for atrial natriuretic peptide have been identified in membrane of the phaeochromocytoma cell line PC12. Scatchard analysis of binding studies revealed a Kd of 794 pM and a density (Bmax) of 254 fmol/mg protein. Hormones unrelated to ANP such as angiotensin II, bradykinin and arginine-8-vasopressin did not complete for the binding sites. Of the ANP-related peptides which competed for the binding sites, the following order of affinity was established; rANP (8-33) greater than rANP (28 amino acid) greater than rat atrial peptide fragment (13-28) greater than a-hANP (28 amino acid) greater than atrial peptide fragment (1-11) greater than atriopeptin I.     

Comparison of binding and cyclic GMP accumulation by atrial natriuretic peptides in endothelial cells

Rat 125I-labeled atrial natriuretic factor (ANF (8-33)) was used to identify ANF receptors on cultured bovine aortic endothelial cells. Specific binding of 125I-ANF at 37 degrees C to confluent endothelial cells was saturable and of high affinity. Scatchard analysis of the equilibrium binding data indicated that endothelial cells contain a single class of binding sites with a Kd of 0.1 +/- 0.01 nM. This particular clone of endothelial cells had 16000 +/- 1300 receptors per cell. The order of potency for competing with 125I-ANF binding was human atrial natriuretic peptide (hANP) = atrial natriuretic factor (ANF (8-33)) greater than atriopeptin II greater than atriopeptin III greater than atriopeptin. The weakest competitor, atriopeptin I, had a K1 of 0.45 nM, which was only 6-fold higher than the K1 for hANP and ANF (8-33). ANF (8-33) and hANP in the presence of 0.5 mM isobutylmethyl-xanthine produced a 15-20-fold increase in cyclic GMP content at 10 pM and a maximal 500-fold elevation of cyclic GMP at 10 nM. The concentrations required to elicit a half-maximal increase in cyclic GMP for hANP, ANF (8-33), atriopeptin I, atriopeptin II and atriopeptin III were 0.30, 0.35, greater than 500, 4.0 and 5.0 nM, respectively. Although atriopeptin I acted as a partial agonist, it was unable to antagonize the effect of ANF (8-33) on cyclic GMP formation. These findings suggest that endothelial cells have multiple and functionally distinct ANF-binding sites.     

Identification in rat atrial tissue of multiple forms of natriuretic polypeptides of about 3,000 daltons

Rat atrial natriuretic peptides of relatively low molecular weight have been isolated from the alpha-component of rectum relaxant activity corresponding to about 3,000 daltons, which was obtained as a side fraction in our previous isolation of beta-rat atrial natriuretic polypeptide (beta- rANP ). In contrast to the same fraction from human atria, the rat atrial alpha-component was found to contain six or more distinct but related peptides, eliciting a potent natriuretic activity. Six of them (B-II, C, D, E, B-I and A), containing 35, 33, 32, 31, 28 and 25 amino acid residues, respectively, have been purified to homogeneity and sequenced. All these peptides were found to correspond to the C-terminal sequence of beta- rANP composed of 48 residues, with varying N-terminal elongations. This indicates that these peptides are derived from beta- rANP . Peptide B-I, composed of 28 residues, is identical to alpha-human atrial polypeptide(alpha- hANP ), with a single replacement of Ile for Met at position 12.     

Atrial natriuretic peptides elevate cyclic GMP levels in primary cultures of rat ependymal cells

The aim of this study was to examine the effect of atrial natriuretic peptides on primary cultures of ependymal cells, as measured by changes in intracellular levels of cyclic GMP. Incubation of ependymal cells with rat atrial natriuretic peptide-(1-28) (rANP) elicited a 30-fold increase in ependymal cGMP content within 1 min and more than a 100-fold increase within 10 min to a plateau value of approximately 30 pmol/mg protein. The C-type natriuretic peptide (CNP) elicited a similar increase in cGMP levels; however the maximal effect was observed within 1 min and the levels subsequently dropped by 90% to a low plateau within 10 min. A comparison of the concentration-response curves for rANP, human ANP-(1-28) (hANP) and CNP showed that rANP, hANP and CNP had similar effects, with regards to elevation of cGMP levels at high concentrations, but with differing EC50 values. These results demonstrate the presence of a heterogenous population of functional ANP receptors in cultured ependymal cells suggesting that ANP may regulate specific ependymal cell activity.     

In vivo regulation of adrenocorticotrophin secretion in the immature ovine fetus. Modulation by ovine corticotrophin releasing hormone and arginine vasopressin

This study investigates the in vivo regulation of ACTH secretion in the immature ovine fetus by AVP and oCRH. Previously we have demonstrated that whilst AVP-containing neurones are present from 42 days, oCRH-containing neurones cannot be detected in the fetal paraventricular nucleus or median eminence until after 90 or 100 days respectively. In acutely exteriorized fetuses aged between 64-90 days (n = 5), a haemorrhagic stress elicited a significant increase (P less than 0.01) in ACTH values. There was also a significant correlation between plasma ACTH and AVP concentrations in these fetuses. In chronically cannulated fetuses less than 100 days (n = 6) injection of AVP (200 ng) significantly elevated fetal plasma ACTH values at 10 min (P less than 0.01) post injection. Simultaneous injection of AVP (200 ng) and oCRH (10 micrograms) into these fetuses produced a plasma ACTH value that was significantly greater at 10 min (P less than 0.05) than the summed response obtained with separate injection of oCRH and AVP. When AVP and oCRH were injection in equimolar amounts to fetuses between 101-118 days, AVP (2 micrograms) was found to have a greater effect on ACTH than was oCRH (2 micrograms). Pretreatment with a specific vascular antagonist of AVP-d(CH2)5Tyr(Me)AVP failed to significantly inhibit the increase in mean arterial pressure associated with AVP (2 micrograms) injection but partially antagonized (P = 0.04) the decrease in fetal heart rate. The antagonist however completely abolished any effect of AVP on fetal plasma ACTH values. This study suggests that AVP most likely acts through receptors in the fetal pituitary with V1 characteristics.     

Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.     

中枢精氨酸加压素在大鼠促肾上腺皮质激素释放激素释放激素引起发热机制中的作用

实验对大鼠进行第三脑室和脑腹中隔区插管,用数字体温计测量大鼠的结肠温度,用放射免疫分析法测定脑中隔区精氨酸加压素（arginine vasopressin,AVP）含量,观察脑中隔区AVP在大鼠促肾上腺皮质激素释放激素（corticotrophin releasing hormone,CRH）性发热机制中的作用。结果发现：脑室注射CRH（5.0μg）引起大鼠结肠温度明显升高,同时明显增高脑中隔区AVP的含量。脑腹中隔区注射AVP V1受体拮抗剂本身并不导致大鼠结肠温度明显改变,但能显著增强脑室注射CRH引起的发热反应。而且,腹中隔区注射AVP显著抑制大鼠CRH性发热。结果提示：发热时CRH是引起脑腹中隔区AVP释放的因素之一,脑腹中隔区内源性AVP抑制中枢注射CRH引起的体温升高。     

心钠素对卒中易感型自发性高血压大鼠血浆和脑组织加压素的影响

为研究心钠素(ANF)和精氨酸加压素(AVP)的相互作用在原发性高血压发病中的意义,对卒中易感型自发性高血压大鼠(SHRsp)和对照大鼠(WKY)侧脑室(icv)或静脉(iv)注射人ANF-(99-126)观察其对血浆、下丘脑和垂体AVP含量以及平均动脉压(MAP)和尿量(UV)、尿钠(U_(Na)V)排泌的影响。静脉注射ANF后10min,SHRsp和WKY大鼠的MAP分别下降9.4％和12.2％(P<0.05),UV分别增加9和20倍(P<0.01),U_(Na)V增加16和29倍(P<0.01)。侧脑室注射ANF对两种大鼠的MAP、UV和U_(Na)V排泌均无明显作用。静脉或侧脑室注射ANF均使两种大鼠的血浆AVP水平明显下降,其中SHRsp的血浆AVP浓度下降程度(iv,-58％;icv,-31％)弱于WKY大鼠(iv,-80％;icv,-65％),下丘脑AVP含量在两种大鼠中都明显增加,而垂体AVP含量无明显变化。 结果表明,人ANF-(99-126)有明显的抑制AVP释放和降压、利尿、利纳作用,而SHRsp对这些作用的敏感性都降低,提示SHRsp对ABF的反应减弱可能在自发性高血压大鼠的发病中具有一定的意义。     

Modulation of insulin and glucagon secretion from the perfused rat pancreas by the neurohypophysial hormones and by desamino-D-arginine vasopressin (DDAVP)

Marked stimulation of glucagon release and modest stimulation of insulin release were observed during in situ perfusion of the rat pancreas with AVP or OT. Glucagon release in response to AVP or OT (200 pg/ml) gradually increased over a 45 min perfusion period reaching maxima of 500% and 300% of the pre-stimulatory levels, respectively. Insulin release transiently increased by 100%. In each case release rates returned to control values immediately after withdrawal of the peptides. Total glucagon release was concentration dependent and linear from 20 pg to 20 ng AVP or OT/ml (r greater than .97). Pancreatic response to DDAVP perfused at 20 ng/ml was virtually indistinguishable from that induced by AVP at 200 pg/ml. This demonstration of a glucagonotrophic action of the neurohypophysial hormones in the in situ perfused rat pancreas confirms earlier studies using isolated islets and bolus IV injection.     

Central vasopressin in the modulation of catecholamine release in conscious rats

Neurons containing arginine vasopressin (AVP) have been shown to project from the paraventricular nucleus of the hypothalamus to the nucleus tractus solitarius (NTS) in the medulla. We investigated whether AVP acts in brain stem regions to influence sympathoadrenal outflow. Cannulae were implanted into the fourth ventricle of rats 7 days prior to the experiment. The effects of intracerebroventricular (icv) injections of AVP, the vehicle, and AVP antagonist, d(CH2)5Tyr(Me)AVP, on mean arterial pressure (MAP) and plasma noradrenaline (NA) and adrenaline (A) levels were determined in conscious unrestrained rats. Injections of AVP (icv, 23 and 73 ng/kg) but not the vehicle increased MAP and plasma NA and A levels. In contrast, iv injection of AVP increased MAP but decreased plasma concentrations of A and NA. The pressor response to icv injection of AVP was abolished by prior icv injection of AVP antagonist. Injection of AVP antagonist (icv, 0.5 and 1.5 microgram/kg) had no effect on MAP or plasma NA or A levels. These results show that centrally injected AVP activates sympathoadrenal outflow, possibly via an inhibition of baroreceptor reflexes. Since centrally administered AVP antagonist did not influence MAP or plasma NA or A levels, it appears that endogenously released AVP does not have a tonic influence on central cardiovascular reflex system in conscious, unrestrained rats.     

Intraventricular administration of arginine vasopressin suppresses prolactin release via a dopaminergic mechanism

The present study was conducted to determine the effects of intracerebroventricular administration of arginine vasopressin (AVP) on the preovulatory prolactin (PRL) surge. Hourly injections of 1 or 5 micrograms AVP from 1200 to 1700 hr on proestrus prevented increases in plasma PRL levels that afternoon. However, following cessation of AVP treatment, a marked increase in PRL levels occurred between 1830 and 2030 hr. This "rebound" secretion of PRL was greater in rats given 5 micrograms AVP than in rats given the lower dose. The suppression of PRL release by AVP appears to be mediated by dopamine since 5 micrograms AVP failed to inhibit PRL release in animals pretreated with the dopamine antagonist domperidone. Interestingly, under these conditions, AVP increased PRL release compared to levels observed in saline-treated rats. In addition to suppressing PRL release, AVP exerted a dose-dependent inhibition of preovulatory LH release. The results suggest a possible interaction between AVP and dopamine in controlling PRL release which likely takes place within the median eminence.     

血管紧张素Ⅱ在紧张应激引起大鼠血压升高中的作用

实验在雄性Sprague Dawley大鼠上进行。实验动物被随机分为对照组、应激组和应激 腹腔注射卡托普利 (captopril)组。应激组大鼠每天给予电击足底结合噪声的应激刺激 ,每日 2次 ,每次 2h ,连续 15d ;应激 ipcaptopril组大鼠在给予应激刺激期间 ,经腹腔内注射captopril 5 0mg/kg d。实验结果观察到 ,15d后 ,三组大鼠平均尾动脉收缩压分别为 :对照组 16 32± 0 5 5kPa (n =7) ,应激组 19 75± 1 0kPa (n =8) ,应激 ipcaptopril组17 6 9± 1 0 7kPa (n =8)。应激 ipcaptopril组大鼠的尾动脉收缩压较对照组动物有显著升高 (P <0 0 5 ) ,但又显著低于应激组大鼠 (P <0 0 5 ) ;同时 ,三组大鼠下丘脑组织中AVP mRNA水平分别为 :对照组 7332 6 6± 5 2 2 6 5 (n =6 ) ;应激组 12 990 33± 15 33 5 8(n =6 ) ,应激 ipcaptopril组 10 6 15 5± 1410 49(n =6 )。应激 ipcaptopril组大鼠下丘脑组织中AVP mRNA水平较对照组有显著升高 (P <0 0 0 1) ,但又显著低于单纯应激组大鼠 (P <0 0 5 )。统计结果显示 :各组大鼠下丘脑组织中AVP mRNA水平与血压之间存在正相关关系 (P <0 0 0 1)。对照组大鼠在侧脑室注射 (icv)选择性血管升压素 (AVP)V1受体拮抗剂d(CH2 ) 5Tyr(Me)AVP 0 3μg后 ,其平均动脉压 (     

Angiotensin II increases ACTH release in the absence of endogenous arginine-vasopressin

Recent studies from our laboratory indicate a primary central site of action of Angiotensin II (AII) to release ACTH. The present studies were designed to test whether AII is able to release ACTH in vivo in a similar fashion in intact, cannulated, freely moving Long-Evans (LE) or in vasopressin (AVP)-deficient, Brattleboro (DI) female rats. The in vivo response to AII was compared with that elicited by synthetic CRF. AII injected i.v. (0.4 or 2 micrograms/100 g BW) induced a significant, dose-related increase in plasma ACTH values 5 and 15 min after injection, in both LE and DI rats. CRF given to LE and DI rats at 0.4 micrograms/100 g BW elicited a larger increase in ACTH plasma values than a similar dose of AII, 5 or 15 min after the injection. Moreover, ACTH levels after CRF in DI rats were significantly greater than those obtained in LE controls. In vitro studies using dispersed anterior pituitary cells indicate that the response of cells from either LE or DI rats to AII or AVP (both at 10(-9) and 10(-8)M) was similar. Cells from DI donors were hyperresponsive to CRF (2 X 10(-11) and 10(-10)M) in terms of ACTH release when compared with the response of cells from LE rats. The present results suggest that the presence of AVP is not essential to mediate the central response to AII and that AII may act centrally to stimulate CRF release from the hypothalamus in vivo, which would then enhance ACTH output. The results in the DI rat indicate that the increased response to CRF may be an important compensatory mechanism involved in the regulation of adrenocortical function in the DI rat.     

Autoradiographic discrimination of brain and atrial natriuretic peptide-binding sites in the rat kidney.

Brain (BNP) and atrial natriuretic peptides (ANP) have been identified which may represent endogenous agonists of kidney receptor subtypes. Quantitative in vitro autoradiography was used to investigate the regional distribution of receptor subpopulations and the competitive inhibition of 125I porcine BNP1-26 (pBNP1-26) and 125I rat alpha-ANP1-28 (rANP1-28) renal binding sites. Specific, high affinity binding (Kd 0.2-1.37 nM range) was localized to glomeruli, inner medulla, interlobar and arcuate arteries, vasa recta bundles, and smooth muscle in the renal pelvis. pBNP1-26 competed for the same sites as rANP1-28 but displayed a lower potency and was less selective for nonclearance sites. Clearance binding sites were discriminated by competitive inhibition with C-ANP4-23 and comprised some 65% of glomerular sites as well as the vast majority of sites in the renal pelvis. Nonclearance sites predominated in the inner medulla and intrarenal arteries. C-terminal changes in amino acid sequence induced a significant loss of inhibitory potency. Immunohistochemical studies identified a distinct population of BNP-like immunoreactive renal nerve fibers, associated with intra-renal arteries. Circulating natriuretic peptides and BNP sequences derived from renal nerves may influence renal function by interacting with specific receptor subpopulations in the kidney.     

Central nervous system control of airway tone in guinea pigs: the role of histamine

The central nervous system (CNS) plays an important role in the reflex control of bronchomotor tone, but the relevant neurotransmitters and neuromodulators have not been identified. In this study we have investigated the effect of histamine. Anesthetized male guinea pigs were prepared with a chronically implanted intracerebroventricular (icv) cannula and instrumented for the measurement of pulmonary resistance (RL), dynamic lung compliance (Cdyn), tidal volume (VT), respiratory rate (f), blood pressure (BP), and heart rate (HR). Administration of histamine (2-30 micrograms) icv caused a significant (P less than 0.05) reduction of Cdyn with no change in RL, VT, and f. At a dose of 100 micrograms icv, histamine caused an increase in RL (202 +/- 78%), a reduction of Cdyn (77 +/- 9%), an increase in f (181 +/- 64%), and a reduction of VT (53 +/- 18%). There were no changes in BP and HR after 100 micrograms of icv histamine. In contrast, intravenous administration of histamine (0.1-2 micrograms/kg) caused a dose-dependent decrease in Cdyn and increase in RL that was associated with tachypnea at each bronchoconstrictor dose. Intravenous histamine (2 micrograms/kg) produced a fall in BP and an increase in HR. The bronchoconstrictor responses to icv histamine were completely blocked by vagotomy and significantly reduced by atropine (0.1 mg/kg iv), whereas vagotomy and atropine did not block the bronchospasm due to intravenous histamine. Additional studies indicated that the pulmonary responses due to icv histamine (100 micrograms) were blocked by pretreatment with the H1-antagonist chlorpheniramine (1 and 10 micrograms, icv). These data indicate that histamine may serve a CNS neurotransmitter function in reflex bronchoconstriction in guinea pigs.     

Identification of an atrial natriuretic peptide specific receptor in human kidney

A specific receptor for human atrial natriuretic peptide (h-ANP) was identified in the human kidney using the radioligand binding assay. Samples were prepared from non-malignant renal tissues obtained at nephrectomy of patients with renal carcinoma. Binding studies using [125I]hANP were performed at 0 degree C for 20 minutes and terminated by a rapid filtration technique. Scatchard plot analysis revealed [125I]hANP bound to a single class of binding site (Kd = 0.4 +/- 0.2 nM) with a density of 16 +/- 4 fmol/mg protein in the renal cortex (n = 7). The binding was rapid and maximal binding was obtained within 20 minutes after the start of incubation. Radioligand displacement was observed in a dose dependent fashion when cold hANP was entered into the reaction mixture. However, unrelated agents, such as angiotensin II or 1-epinephrine, did not affect the binding. This is the first time characterization of the hANP receptor in the human kidney has been conducted using a Scatchard plot analysis.     

Galanin: structural requirements for binding and signal transduction in RINm5F insulinoma cells.

Receptors for galanin, a neuropeptide inhibiting insulin release, have been described on RINm5F insulinoma cells. To characterize structural requirements for binding and biological activity of galanin, we studied binding and inhibition of hormone stimulated intracellular cAMP-production of N-terminal galanin fragments and -analogues in RINm5F cells. Half-maximal binding and potency were the same for all peptides used. Active peptides had the following rank of potency: galanin = galanin(1-22(23)Cys) greater than galanin(1-29(4)NLe) greater than galanin(1-18) greater than galanin(1-29(7)DAla) greater than galanin(1-29(2)DTrp4NLe7DAla) greater than galanin(1-29(2)DTrp). Galanin(3-29) was inactive. Therefore the first two amino acids of the galanin molecule with the indole side chain of the tryptophane residue in the right steric position are crucial for receptor binding.