Differential distribution of two molecular forms of gonadotropin-releasing hormone in discrete brain areas of goldfish (Carassius auratus)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were identified in the extracts of various brain areas, spinal cord and pituitary in female and male goldfish and had chromatographic and immunological properties similar to [His5, Trp7, Tyr8]-GnRH (cGnRH-II) and [Trp7,Leu8]-GnRH (sGnRH). Radioimmunoassay using different GnRH antisera after high pressure liquid chromatography did not reveal significant peaks of mammalian GnRH, [Gln8]-GnRH and [Tyr3,Leu5,Glu6,Trp7,Lys8]-GnRH in the brain extracts. The proportion of cGnRH-II-like immunoactivity to sGnRH-like immunoactivity was higher in the caudal brain areas compared to the rostral areas. The differential distribution of two GnRH forms suggest that the different GnRH forms may have different physiological functions.     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary.

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of gonadotropin-releasing hormone (GnRH) receptors in the ovary of common carp (Cyprinus carpio).

Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 degrees C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0-40.0 micrograms membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10(-6) M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-PTrp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of His5,Trp7,Tyr8-GnRH (chicken GnRH II) in amphibian brain

GnRH immunoreactive and bioactive peptides in Xenopus laevis brain extract were investigated by high performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera raised against GnRH (mammalian), His5,Trp7,Tyr8-GnRH (chicken II) and Tyr3,Leu5,Glu6,Trp7,Lys8-GnRH (lamprey), and by assessment of biological activity. Two immunoreactive peptides eluted in the same positions as GnRH and His5,Trp7,Tyr8-GnRH respectively in HPLC systems which were specifically designed to separate four known natural vertebrate GnRHs (mammalian, chicken I and II, salmon). The immunological properties of these two immunoreactive peaks, determined by relative interaction with three region-specific antisera raised against mammalian GnRH and two specific His5,Trp7,Tyr8-GnRH antisera, were identical to those of GnRH and His5,Trp7,Tyr8-GnRH. The immunoreactive peak co-eluting with His5,Trp7,Tyr8-GnRH represented approximately one-third of the total brain GnRH. Both immunoreactive peaks stimulated luteinizing hormone (LH) release in a chicken dispersed pituitary cell bioassay, and the amounts of LH release stimulated by the two peaks were appropriate for these peaks being GnRH and His5,Trp7,Tyr8-GnRH. A small hydrophobic peak with GnRH immunoreactivity eluted in the same position as Trp7,Leu8-GnRH (salmon), while Gln8-GnRH (chicken I) and lamprey GnRH were not detected. Two additional rather hydrophilic peptides cross-reacted with a COOH-terminus-directed antiserum and had LH-releasing activity. LH-releasing activity was also detected in hydrophobic HPLC fractions. In summary, these data provide evidence for the presence of both GnRH and a second peptide with properties identical to His5,Trp7,Tyr8-GnRH in X. laevis brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist.

Gonadotropin-releasing hormone (GnRH) stimulates release of gonadotropin hormone (GTH) through interaction with high affinity receptors in the goldfish pituitary. In the present study, we investigated desensitization of two native GnRH peptides, [Trp7, Leu8]-GnRH (sGnRH) and [His5, Trp7, Tyr8]-GnRH (cGnRH-II), using superfused fragments of goldfish pituitary in vitro. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses given every 60 min) resulted in dose-dependent secretion of GTH from the goldfish pituitary; cGnRH-II had a greater GTH release potency and displayed a greater receptor binding affinity than sGnRH. Both sGnRH and cGnRH-II-induced GTH release were partially inhibited by concomitant treatment with either [D-Phe2, Pro3, D-Phe6]-GnRH or [D-pGlu1, D-Phe2, D-Trp3.6]-GnRH. These antagonists had greater receptor binding affinities than the native peptides, with no stimulatory action on GTH release in the absence of the GnRH agonists. Continuous treatment with either sGnRH or cGnRH-II (10(-7) M), rapidly desensitized pituitary GTH release in a biphasic fashion; initially there was a rapid increase in GTH release of approximately 10-20-fold (phase 1), followed by a sharp decline in GTH release, reaching a stable concentration 2-3-fold above the basal level (phase 2). Further stimulation of the pituitaries with sGnRH or cGnRH-II (10(-7) M) (second treatment) after 60 min recovery resulted in a significantly lower sGnRH or cGnRH-II-induced GTH release compared to that observed during the initial treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of pituitary gonadotropin-releasing hormone receptors in goldfish (Carassius auratus)

Receptors for GnRH were labeled by use of an iodinated (125I) photoreactive GnRH derivative [D-Lys6-azidobenzoyl]-GnRH. This derivative was found to bind to two classes of GnRH binding sites: high-affinity/low-capacity sites and low-affinity/high-capacity sites. The binding affinity of [D-Lys6-azidobenzoyl]-GnRH was found to be greater than that of D-Lys6-GnRH, but lower than a superactive fish GnRH agonist [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH (sGnRH-A). Analysis of the photoaffinity-labeled goldfish pituitary GnRH receptors by SDS-PAGE and autoradiography indicated the presence of three labeled proteins displaceable by unlabeled sGnRH-A. The first and the most prominently labeled band was a 71,000-Mr protein, the second a 51,000-Mr protein, and the third a minor band of 130,000 Mr. Displacement characteristics of the 71,000- and 130,000-Mr bands were consistent with those of the low-affinity binding sites; displacement of the iodinated ligand from these proteins was achieved only in the presence of 10(-6) M sGnRH-A. The 51,000-Mr band had characteristics similar to those of the high-affinity site; displacement of the labeled ligand was achieved in the presence of 10(-9) M sGnRH-A. These findings provide for the first time some biochemical characterizations of pituitary GnRH receptors in a nonmammalian vertebrate.     

Effects of exercise on plasma catecholamine levels in the toad, Bufo paracnemis: role of the adrenals and neural control

Resting plasma epinephrine (E) and norepinephrine (N) concentrations for intact toads (Bufo paracnemis) were 5.57+/-1.0 and 0.88+/-0.38 ng/ml, respectively. Exercise induced a significant increase in heart rate, blood pressure and plasma epinephrine (about 4.3 times), whereas norepinephrine remained unchanged. The resting [E]/[N] ratio was 6.3 and increased to 32.9 during exercise. Adrenal denervation did not alter the basal plasma catecholamine or norepinephrine levels after exercise, but prevented the increase in epinephrine during exercise, suggesting that in the intact toad this increase is due to adrenal secretion whereas resting norepinephrine may be liberated by extra-adrenal chromaffin tissues. This also suggests that the adrenal glands can release selectively the two catecholamines. The increases in heart rate and blood pressure in denervated toads were not significantly different from those of intact animals, suggesting that during exercise the sympathetic nerves play the main role in inducing cardiovascular responses. Spinal transection induced a significant increase in basal norepinephrine levels, which remained elevated after exercise. Since spinal toads are unable to perform spontaneous movements it is possible that this increase may be caused by this stressful condition. The increases in heart rate and blood pressure observed in spinal toads during exercise may be due to direct mechanical effects of venous return on the heart.     

Chimeric analogues of vertebrate gonadotropin-releasing hormones comprising substitutions of the variant amino acids in positions 5, 7, and 8. Characterization of requirements for receptor binding and gonadotropin release in mammalian and avian pituitary gonadotropes

Variant forms of mammalian gonadotropin-releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH2) are present in chicken ([Gln8] GnRH and [His5, Trp7, Tyr8]GnRH), salmon ([Trp7, Leu8]GnRH), and lamprey ([Tyr3, Leu5, Glu6, Trp7, Lys8] GnRH). To delineate the functional importance of the variant amino acids in positions 5, 7, and 8, the natural peptides and chimeric analogues were tested for gonadotropin-releasing activity and receptor-binding activity in rat, sheep, and chicken pituitaries. The results demonstrate that (i) the mammalian receptor has a high fidelity for Arg8 while the chicken receptor is less discriminatory and accepts basic or neutral amino acids in this position. Arg8 may contribute to conformational stabilization, and conformational constraint with D-Trp6 restored activity to analogues lacking Arg8 in the mammalian systems. D-Trp6 incorporation did not generally enhance activity in the chicken pituitary. (ii) His5 accompanying Arg8 in analogues markedly diminished activity in the chicken while gonadotropin-releasing activity was retained in the sheep pituitary. Receptor-binding activity was increased in the sheep indicating an uncoupling of receptor occupancy and activation. (iii) Substitution in position 7 is tolerated by the mammalian and chicken receptor. With Trp7-substituted analogues receptor-binding activity was relatively lower than gonadotropin-releasing activity in the sheep pituitary, suggesting an enhanced receptor activation by these analogues or the existence of different GnRH receptors.     

Identification of Gln8-GnRH and His5,Trp7,Tyr8-GnRH in the hypothalamus and extrahypothalamic brain of the ostrich (Struthio camelus)

Gonadotropin-releasing hormone (GnRH) molecular forms were studied in extracts of ostrich hypothalamus and extrahypothalamic brain using high performance liquid chromatography, radioimmunoassay with region-specific antisera and assessment of luteinizing hormone (LH)-releasing activity using chicken dispersed pituitary cells. Two molecular forms of GnRH with chromatographic, immunological and biological properties identical to those of Gln8-GnRH and His5,Trp7,Tyr8-GnRH were demonstrated in both the hypothalamic and extrahypothalamic brain extracts. A greater proportion of His5,Trp7,Tyr8-GnRH was present in the hypothalamus than in extrahypothalamic brain. It is likely that these two forms of GnRH are present in all bird species, since the chicken and the ostrich have evolved separately.     

Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

Peptide N-alkylamides by solid phase synthesis

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH2CH3, NH2CH2CH3, NH2CH2CF3) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [DGlu6, Pro9-NHCH2CH3]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [DGlu6]-GnRH. Other analogs including [DTrp6, Pro9-NHCH2CH3]-GnRH, [DAla6, Pro9-NHCH2CF3]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the CO group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.     

Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi.

Seven new antagonists of bombesin (Bn)/gastrin-releasing peptide (GRP) containing C-terminal Trp or Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-carboxylic acid) in a reduced peptide bond were synthesized by solid phase methods and evaluated biologically. The reduced bond in four [Leu13 psi(CH2NH)Trp14]Bn(6-14) analogs was formed by reductive alkylation at the dipeptide stage. In the case of three [Leu13 psi(CH2N)Tpi14]Bn(6-14) analogs, the Trp dipeptide with reduced bond was reacted with formaldehyde to form the corresponding Tpi derivative. These Tpi-containing analogs have a new reduced bond which is structurally more constrained. Leu13 psi(CH2N)Tpi14 analogs inhibit [125I][Tyr4]bombesin binding to Swiss 3T3 cells with IC50 values of 2-4 nM, compared to 5-10 nM for Leu13 psi(CH2NH)Trp14 analogs. Leu13 psi(CH2N)Tpi14 analogs are also more potent than Leu13 psi(CH2NH)Trp14 analogs in growth inhibition studies using Swiss 3T3 cells. The two best bombesin antagonists of this series, [D-Trp6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3415) and [Tpi6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3440), inhibited GRP-stimulated growth of Swiss 3T3 cells with IC50 values less than 1 nM. RC-3440 was also active in vivo, suppressing GRP(14-27)-stimulated serum gastrin secretion in rats. Bombesin/GRP antagonists, such as RC-3440, containing the new reduced bond (CH2N) reported herein are very potent.     

Hypotensive mechanism of [Leu13]motilin in dogs in vivo and in vitro.

The effects of [Leu13]motilin were examined in vivo after its intravenous administration into anesthetized dogs and in vitro with isolated preparations of canine mesenteric artery. [Leu13]Motilin (0.1-10 nmol x kg(-1), i.v.) induced both strong and clustered phasic contractions in the gastric antrum and duodenum. At doses of over 1 nmol x kg(-1), [Leu13]motilin also produced transient decreases in arterial blood pressure, left ventricular pressure, maximum rate of rise of left ventricular pressure, and total peripheral resistance, and an increase in aortic blood flow and heart rate. A selective motilin antagonist, GM-109 (Phe-cyclo[Lys-Tyr(3-tBu)-betaAla] trifluoroacetate), completely abolished the gastric antrum and duodenal motor responses induced by [Leu13]motilin. In contrast, hypotension induced by [Leu13]motilin (1 nmol x kg(-1)) was unchanged in the presence of GM-109. In isolated mesenteric artery preparations precontracted with U-46619 (10(-7) M), [Leu13]motilin (10(-8)-10(-5) M) induced an endothelium-dependent relaxation, and this was inhibited by a pretreatment with N(omega)-nitro-L-arginine, a competitive inhibitor of NO synthase (10(-4) M). A high dose (10(-4) M) of GM-109 slightly decreased [Leu13]motilin-induced relaxation, and shifted the concentration-response curve of [Leu13]motilin to the right. However, the pA2 value (4.09) of GM-109 for [Leu13]motilin in the present study was conspicuously lower than that previously demonstrated in the rabbit duodenum (7.37). These results suggest that [Leu13]motilin induces hypotension via the endothelial NO-dependent relaxation mechanism and not through the receptor type that causes upper gastrointestinal contractions.     

Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl [2,4(5)-NAPS] group into amino acid residues at positions 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp3 by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The [D-Lys(2,4-NAPS)]6 analogue was a very potent agonist that, after covalent attachment by photoaffinity labeling, caused prolonged LH secretion at a submaximal rate. [Orn(2,4-NAPS)]8-GnRH, a full agonist with a relative potency of 7% of GnRH, after photoaffinity labeling caused prolonged maximal LH release from cultured pituitary cells. In contrast, [Orn(2,5-NAPS)]8-GnRH, although being equipotent with the 2,4-NAPS isomer in terms of LH releasing ability, was unable to cause prolonged LH release after photoaffinity labeling. Thus, [Orn(2,4-NAPS)]8-GnRH is a very effective photolabeling ligand of the functionally significant pituitary GnRH receptor. Based on this compound, a pituitary peptidase resistant derivative, D-Phe6,[Orn(2,4-NAPS)]8-GnRH-(1-9)-ethylamide, was synthesized. This derivative showed high-affinity binding to pituitary membranes with a Kd comparable to those of other GnRH analogues. A radioiodinated form of this peptide was used for pituitary GnRH-receptor labeling. This derivative labeled 59- and 57-kDa proteins in rat and 58- and 56-kDa proteins in bovine pituitary membrane preparations, respectively. This peptide also labeled pituitary GnRH receptors in the solubilized state and therefore appears to be a suitable ligand for the isolation and further characterization of the receptor.     

Interaction of substance P antagonists with substance P receptors on dispersed pancreatic acini

In the present study we examined the abilities of three analogs of substance P, [D-Pro2-, D-Phe7-, D-Trp9]-substance P, [D-Pro2-, D- Trp7 ,9]-substance P and [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to alter substance P-induced changes in pancreatic acinar cell function and to occupy substance P receptors. At 30 microM, each analog of substance P lacked agonist activity and inhibited amylase secretion stimulated by substance P receptor agonists. The inhibition was reversible and specific for peptides that interact with substance P receptors (physalaemin, substance P, eledoisin, kassinin ). The analogs of substance P did not inhibit the actions of cholecystokinin, caerulein, gastrin, carbamylcholine, secretin, vasoactive intestinal peptide, PHI, ionophore A23187 or 8Br -cAMP. At high concentrations, [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P, but not [D-Pro2-, D- Trp7 ,9]-substance P or [D-Pro2-, D-Phe7-, D-Trp9]-substance P, caused a small but significant inhibition of bombesin-stimulated amylase release. For each analog of substance P, the inhibition was competitive in nature in that there was a rightward shift of the dose-response curve for physalaemin-stimulated amylase secretion with no change in efficacy. From Schild plots of the ability of [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to inhibit either substance p- or physalaemin-stimulated amylase release, the slopes were not different from unity. For each analog of substance P, there was a close correlation between its ability to inhibit substance P- or physalaemin-stimulated amylase release and its ability to inhibit binding of 125I-labeled substance P or 125I-labeled physalaemin. [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P was 2-fold more potent than [D-Pro2-, D- Trp7 ,9]-substance P which was 4-fold more potent than [D-Pro2-, D-Phe7-, D-Trp9]-substance P, (i.e., pA2 6.1, 5.9, and 5.2, respectively). For each analog, the dose-response curve for its ability to inhibit physalaemin-stimulated amylase release was superimpossible on the dose-response curve for its ability to inhibit binding of 125I-labeled physalaemin. These results indicate that each of these analogs of substance P is a specific competitive inhibitor of the action of the substance P on dispersed acini from guinea-pig pancreas, and that their abilities to inhibit substance P-induced changes in acinar cell function can be accounted for by their abilities to occupy the substance P receptor.     

Effect of physical training on the capacity to secrete epinephrine

Epinephrine responses to hypoglycemia and to identical relative work loads have been shown to be higher in endurance-trained athletes than in untrained subjects. To test the hypothesis that training increases the adrenal medullary secretory capacity, we studied the effects of glucagon (1 mg/70 kg iv), acute hypercapnia (inspired O2 fraction = 7%), and acute hypobaric hypoxia (inspired Po2 = 87 Torr), respectively, on the epinephrine concentration in arterialized hand vein blood in eight endurance-trained athletes [T, O2 uptake = 66 (62-70) ml.min-1.kg-1] and seven sedentary males [C, O2 uptake = 46 (41-50)]. In response to identical increments in glucagon concentrations, plasma epinephrine increased more in T than in C subjects [0.87 +/- 0.11 vs. 0.38 +/- 0.14 (SE) nmol/l, P less than 0.05]. In response to hypercapnia [arterial PCO2 = 56 +/- 0.7 Torr (T) and 55 +/- 0.4 (C), P greater than 0.05], the increment in epinephrine was significant in T (0.38 +/- 0.11 nmol/l) but not (P less than 0.1) in C subjects (0.22 +/- 0.11). Hypoxia [arterial PO2 = 42 +/- 2 Torr (T) and 41 +/- 2 (C), P greater than 0.05] increased epinephrine in T (0.22 +/- 0.10 nmol/l, P less than 0.05) but not in C subjects (0.01 +/- 0.07). The plasma norepinephrine concentration never changed, whereas heart rate always increased, the increase being higher (P less than 0.05) in T than in C subjects only during hypercapnia. The results indicate that training increases the capacity to secrete epinephrine.     

Cardiovascular responses to catecholamines at 12 degrees C in the American bullfrog (Rana catesbeiana)

The effects of epinephrine, norepinephrine, phenylephrine, and isoproterenol on blood pressure and heart rate were studied in cannulated American bullfrogs, Rana catesbeiana. The bullfrogs were chronically cannulated with a T cannula in the right sciatic artery. In warm-acclimated (22 degrees C) bullfrogs, preinjection mean systemic arterial pressure (SAP) prior to experimental treatment was 13.1 +/- 0.7 mm Hg. Preinjection heart rate was 34.8 +/- 1.8 beats per minute. These parameters were lower in cold-acclimated (12 degrees C) bullfrogs. Cold-acclimated animals had mean SAP values of 8.2 +/- 0.3 mm Hg, and heart rate was 11.1 +/- 1.1 beats per minute. Epinephrine, norepinephrine, and phenylephrine increased blood pressure to an equivalent degree in warm- and cold-acclimated animals. Dose-related decreases in heart rate in response to these catecholamines were observed in warm- but not in cold-acclimated bullfrogs. Warm-acclimated animals were more responsive to isoproterenol from 0.03 micrograms/kg body weight (bw) to 10 micrograms/kg bw than were cold-acclimated animals. The response to isoproterenol was effectively blocked by propranolol (5 mg/kg bw) in both warm- and cold-acclimated animals. Propranolol alone decreased mean SAP in both warm- and cold-acclimated animals, suggesting blockade of endogenous sympathetic activity. Beta receptor response thus appears diminished, but not absent at 12 degrees C. However, the alpha receptors responsible for elevation of blood pressure equally responsive at 12 degrees and 22 degrees C.