Do growth hormone-releasing peptides act as ghrelin secretagogues?

NN703 is an orally active and selective growth hormone secretagogue (GHS) that was derived from growth hormone-releasing peptide-1(GHRP-1) via ipamorelin by a peptidomimetic approach and has now entered into phase II clinical trials. When the disposition in rats of NN703 and GHRP-6 was studied using whole-body autoradiography following administration of an iv dose of radiolabeled material, we found that a substantial amount of these secretagogues accumulate in the glandular part of the stomach. Because this is the site of synthesis and secretion of ghrelin, the endogenous GHS, we investigated the effect of resection of the gastrointestinal (GI) tract on growth hormone (GH) release induced by GHRP-6. This procedure significantly attenuated the GH secretion response by 60-70%. By contrast, the effect of GH-releasing hormone on GH release was not inhibited. The binding of GHRPs to the glandular part of the stomach and the blunted GH response to GHRP-6 following resection of the GI tract suggest a role for ghrelin as a mediator of part of the GH-releasing effect of GHRPs.

Nociceptin is a potent inhibitor of N-type Ca(2+) channels in rat sympathetic ganglion neurons.

Nociceptin, an endogenous agonist of the opioid receptor-like(1) (ORL(1)) receptor, is implicated in a wide range of physiological functions including cardiovascular control. However, the effect of nociceptin on peripheral sympathetic ganglion neurons has not been studied. Whole-cell voltage clamp was used to study Ca(2+) currents on freshly dissociated sympathetic superior cervical ganglion neurons from juvenile rats. Nociceptin (1 microM) caused a fast inhibition of the peak currents by 69+/-3% in all neurons. Strong positive prepulses counteracted the inhibition of the peak current by 64% and no effect of nociceptin was observed when the cells were pre-incubated with Pertussis toxin. The inhibition was reversible and dose-dependent with an EC(50) of 508+/-50 pM. Blockade of N-type channels by 1 microM omega-conotoxin GVIA reduced the peak currents by 83+/-1% and abolished the action of nociceptin. Naloxone could not prevent the inhibition by nociceptin and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin (DAMGO) only depressed a small proportion of the current in 1/7 neurons. These data suggests that nociceptin inhibits transmitter release from sympathetic neurons by a selective blockade of N-type channels, which may be of importance for its depressive effect on the cardiovascular system.

[3H]ac-RYYRWK-NH2, a novel specific radioligand for the nociceptin/orphanin FQ receptor.

The hexapeptide ac-RYYRWK-NH2 has been described as a potent partial agonist at the nociceptin (NC)/orphanin FQ receptor which has no affinity for mu-, kappa- or delta-opioid receptors. However, it is not clear whether ac-RYYRWK-NH2 is truly selective for the NC receptor, and ac-RYYRWK-NH2 has therefore been radiolabelled and characterised in receptor-binding experiments. Saturation experiments with [3H]ac-RYYRWK-NH2 binding to rat cortical membranes revealed a single high affinity site for [3H]ac-RYYRWK-NH2 (Kd=0.071 +/- 0.018 nM; Bmax=22+/-2 fmol/mg protein). Uncoupling of the G-proteins resulted in a significant 45% increase in Kd and no change in Bmax. [3H]ac-RYYRWK-NH2 binding to rat cortical membranes or to membranes from baby hamster kidney cells expressing human orphan opioid receptor-like (ORL1) was displaced by NC and ac-RYYRWK-NH2 to the same extent. The following rank order of potency was observed: ac-RYYRWK-NH2 > [Tyr14]NC-OH = NC-OH = NC-NH2 > NC, H-(1-13)-NH2 > NC(1-12)-NH2 >> NC(1-11)-NH2 and, thus, displayed a typical NC receptor pharmacology. Novel cyclic analogues of ac-RYYRWK-NH2 were prepared but these structures were much less active when compared to ac-RYYRWK-NH2. In vitro receptor autoradiography with [3H]ac-RYYRWK-NH2 to rat brain sections revealed high levels of binding in the cerebral cortex, amygdala, hypothalamus and superior colliculus, but low levels in the cerebellum and striatum. Overall, the regional distribution was very similar to that of [3H]NC. Ac-RYYRWK-NH2 seems indeed to be selective for the NC receptor and [3H]ac-RYYRWK-NH2 is a novel radioligand which may be useful for further exploring the pharmacology and receptor-ligand interaction of the NC receptor.

Possible mechanism of c-fos expression in trigeminal nucleus caudalis following cortical spreading depression.

Cortical spreading depression (CSD) is characterized by a transient, reversible depression of EEG activity which advances across the cortical surface at a velocity of 2-5 mm/min. CSD was originally linked to the aura phase of migraine, but recently also to migraine headache. The theory is that CSD activates meningeal trigeminal C-fibers causing neurogenic inflammation and pain (Moskowitz, M.A., Nozaki, K. and Kraig, R.P., Neocortical spreading depression provokes the expression of c-fos protein-like immunoreactivity within trigeminal nucleus caudalis via trigeminovascular mechanisms, J. Neurosci., 13 (1993) 1167-1177). The present study is an examination of the proposed link between CSD elicited in rats and activation of trigeminal nerve fibers. Multiple CSDs were elicited unilaterally for 1 h by KCl injections (1 M, 5 microliters) into the right hemisphere, while NaCl (1 M, 5 microliters) was injected into the left as control. After an additional 1 h the animals were sacrificed and trigeminal activation assessed by the expression of c-fos in trigeminal nucleus caudalis (TNC) using immunohistochemistry. The correlation between the number of CSDs and the extent of c-fos expression was determined. In addition the effect of sumatriptan (0.3 mg/kg) and morphine (3 mg/kg) given i.v. 30 min before elicitation of CSD was evaluated. CSD caused increased c-fos expression in lamina I and II of TNC where C-fibers, end, the response being greater ipsilaterally. Morphine, but not sumatriptan, reduced c-fos expression in both the ipsilateral and contralateral TNC by 71% (P < 0.05 and P = 0.19, respectively), confirming that nociceptors have been activated. No positive correlation was seen between the number of CSDs and the extent of c-fos expression in TNC. Instead we observed a positive, linear correlation between the number of KCl injections and the extent of c-fos expression in TNC (correlation coefficient r = 0.709, P < 0.05). We suggest that the C-fiber activation observed is caused by hyperosmolar KCl/NaCl and not CSD. Hence, our results do not support the hypothesis of Moskowitz et al. (Moskowitz, M.A., Nozaki, K. and Kraig, R.P., Neocortical spreading depression provokes the expression of c-fos protein-like immunoreactivity within trigeminal nucleus caudalis via trigeminovascular mechanisms, J. Neurosci., 13 (1993) 1167-1177) which links CSD with migraine headache.

Rat gastric relaxation induced by stimulation of endothelin-1 selective receptors.

We have investigated the smooth muscle activity of ET-1 and ET-3 on rat fundus strips in vitro as well as the effects of the peptides on gastric motility in vivo. In the isolated tissue with no precontraction ET-1 and ET-3 were potent spasmogens which produced half maximal contractions at concentrations 4.5 and 8.0 nM, respectively. In contrast, under conditions where the isolated tissue was precontracted to approx. 50% of maximum by prostaglandin E2, ET-1 dose-dependently (5 x 10(-10) - 10(-8) M) and temporarily relaxed the fundus strip, whereas ET-3 further increased the contraction. The relaxing capacity of ET-1 was absent when the tissue was precontracted by potassium yet was resistant to pretreatments with tetrodotoxin, capsaicin, propranolol, indomethacin, NG-methyl-L-arginine or glibenclamide. In addition in vivo ET-1 and ET-3 (less than 1 nmol/kg) showed opposite effects on gastric motility as the former reduced basal tonus and spontaneous activity, whereas the latter increased the motor activity of the gastric ventricle. The results support the notion that ET-1 may induce gastric relaxation by stimulation of selective receptors whereas stimulation of nonselective receptors may promote gastric smooth muscle contraction.

Pharmacological manipulations of anoxia-induced free fatty acid accumulation in the mouse brain.

The accumulation of free fatty acid (FFA) in the brain occurs within minutes of anoxia, induced by exposing mice to a 100% N2 atmosphere. The rate of FFA release is high within the first minute and continues to increase moderately hereafter. FFA is apparently accumulated at the highest concentration in the cerebral hemispheres. The release of FFA can be inhibited partly by CNS depressants like N6-cyclopentyladenosine, pentobarbital, ethanol, or 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3(2H)-one (THIP). Antiadrenergic compounds such as reserpine, clondine, or prazosine were also found to be active. The N2 anoxia was initially and temporarily associated with motor excitation termed fight and flight reaction. This behavior could be reduced by administration of N6-cyclopentyl-adenosine, pentobarbital, ethanol, reserpine, and prazosine, but not by THIP or clonidine. The glutamate antagonist MK-801 inhibited the fight and flight reaction, but did not affect the FFA accumulation. The data are consistent with the view that brain anoxia initially increases FFA by receptor-mediated polyphosphoinositide breakdown and that the alpha-1 adrenergic receptor is one of the receptors involved. The data also indicate that the fight and flight reaction is dissociated from the events that lead to FFA release, and may involve the stimulation of glutaminergic NMDA receptors.

Inhibition by glutamate antagonists, MK-801 and NBQX, of cutaneo-cardiovascular pain reflex in rats.

In urethane-anesthetized rats, xylene applied locally to the skin of the hind paws was shown to induce reflex increases of blood pressure (33%) and heart rate (37%). The blood pressure elevation was dose dependently inhibited by the NMDA antagonist, MK-801 (0.3-1.0 mg/kg i.v.), and by the AMPA (D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid) antagonist, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline 0.1-1.0 mg/kg per min). In contrast, only the latter compound was shown to block dose dependently the observed increase in heart rate. The results suggest that the two glutamate antagonists inhibit nociceptive impulse traffic at distinct anatomical sites and/or by different modes of actions.

Human mononuclear cell chemiluminescence and interleukin-1 release induced by Staphylococcus aureus protein A.

Staphylococcus aureus protein A (SpA) is shown to induce a temporary chemiluminescence (CL)-response of human peripheral blood mononuclear cells. Maximal activity of SpA is observed at 3 micrograms/ml and EC50 is about 1 microgram/ml. The CL response depends on extracellular calcium, and moreover is inhibited by pretreating cells with rabbit-anti-human IgG's. From glass-adhered monocytes, SpA alone (3 micrograms/ml) did not consistently affect the generation of immunoreactive interleukin-1 (IL-1). However, SpA and endotoxin (1 micrograms/ml) showed a marked synergistic effect on IL-1 release. The results suggest that SpA interacts with membrane-bound Ig molecules endowed with receptor functions. The CL response may be envisaged as a result of early transmembrane signalling events presumably taking place in monocytes. Apparently, hereby, the cells become primed for endotoxin-triggered IL-1 release.

[Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist.

Two types of binding sites have previously been described for 36-amino acid neuropeptide Y (NPY), called Y1 and Y2 receptors. Y2 receptors can bind long C-terminal fragments of NPY-e.g., NPY-(13-36)-peptide. In contrast, Y1 receptors have until now only been characterized as NPY receptors that do not bind such fragments. In the present study an NPY analog is presented, [Leu31, Pro34]NPY, which in a series of human neuroblastoma cell lines and on rat PC-12 cells can displace radiolabeled NPY only from cells that express Y1 receptors and not from those expressing Y2 receptors. The radiolabeled analog, [125I-Tyr36] monoiodo-[Leu31, Pro34]NPY, also binds specifically only to cells with Y1 receptors. The binding of this analog to Y1 receptors on human neuroblastoma cells is associated with a transient increase in cytoplasmic free calcium concentrations similar to the response observed with NPY. [Leu31, Pro34]NPY is also active in vivo as it is even more potent than NPY in increasing blood pressure in anesthetized rats. It is concluded that [Leu31, Pro34]NPY is a specific Y1 receptor agonist and that the analog or variants of it can be useful in delineating the physiological importance of Y1 receptors.

Elevated urinary prostaglandin excretion and the effect of indomethacin on renal function in incipient diabetic nephropathy.

We investigated whether the glomerular synthesis of prostaglandins modulates the glomerular filtration rate and urinary albumin excretion in incipient diabetic nephropathy (defined as urinary albumin excretion between 30 and 300 mg/24 h (microalbuminuria) in two out of three sterile ketone-free 24-h urine collections in patients having insulin-dependent diabetes mellitus (IDDM) without hypertension or other kidney disease). The urinary excretion of prostaglandin E2 was significantly elevated in 8 insulin-dependent diabetic patients with incipient nephropathy as compared with 9 normoalbuminuric IDDM patients and 11 healthy controls: 317 (182-1273); 95 (67-225); 132 (54-263) pg/min, respectively (2p less than 0.01). Glomerular filtration rate (single bolus 51Cr-EDTA technique) and albuminuria (radioimmunoassay) were measured twice within 2 weeks in 8 females having IDDM with incipient nephropathy. The study design was a randomized double-blind trial with the patients receiving either indomethacin (150 mg/day) or placebo for 3 days prior to the kidney function studies. Indomethacin treatment induced a significant reduction in urinary prostaglandin E2 excretion (73%) (2p less than 0.01), urinary albumin excretion rate diminished from 207 (63-253) to 87 (49-147) mg/24 h (2p less than 0.01), fractional clearance of albumin declined (70%) (2p less than 0.01). Glomerular filtration rate remained stable (108 (88-133) versus 110 (95-142) ml/min). Blood glucose and blood pressure were comparable during the placebo and indomethacin treatment (12.6 +/- 3 versus 13.4 +/- 5 mmol/l and 122/79 +/- 3/9 versus 122/82 +/- 4/10 mmHg, respectively). Our results suggest that enhanced glomerular synthesis of vasodilating prostaglandins may accelerate microalbuminuria in incipient diabetic nephropathy.

Complement C5a receptor antagonism by protamine and poly-L-Arg on human leukocytes.

It is shown that protamine selectively and dose-dependently inhibits complement C5a-induced leukocyte responses such as histamine release from basophils, chemiluminescence and beta-glucuronidase release from neutrophils. Protamine produces parallel rightward displacements of the C5a dose-response curves. The inhibitory capacity of the polypeptide is reversible and disappears following repeated washing of exposed cells. In neutrophils poly-L-Arg similarly and specifically antagonizes C5a-induced chemiluminescence and enzyme release. This polymer alone, however, degranulates basophils and neutrophils, leading to histamine and enzyme release, respectively. It is concluded that on human neutrophils the arginine-rich polycations protamine and poly-L-Arg exhibit a competitive C5a receptor antagonism. In addition, protamine inhibits the C5a receptors on basophils. It is hypothesized that molecular conformations of the arginine-rich polycations might bind reversibly to, and block negatively charged groups at the C5a-receptor sites.

Endotoxin pretreatment enhances neutrophil FMLP-receptor binding and activity in guinea pigs.

Polymorphonuclear leukocytes isolated from endotoxin pretreated (0.1 mg/kg 24 hours before) guinea pigs are shown to be hypersensitive and hyperresponsive to the formylated bacterial chemoattractant FMLP in vitro. Dose-response curves for chemiluminescence and beta-glucuronidase release are shifted to the left and maxima are increased. In receptor binding studies the FMLP binding capacity is shown to be enhanced in cells from endotoxin pretreated animals. FMLP (0.3 mg/kg) administered intravenously into anaesthetized and artificially ventilated guinea pigs is shown to induce neutropenia and a biphasic rise of the insufflation pressure. This response is exaggerated in endotoxin pretreated animals. The initial elevation of the airway resistance is cyclooxygenase dependent, whereas the following rise is cyclooxygenase independent and parallels the neutropenia. Histologically PMN's are shown to be trapped in the pulmonary capillaries. This is associated with an intraseptal/interstitial edema. The results illustrate a functional synergism between two important bacterial products, endotoxin and FMLP.

Effects of indomethacin on kidney function in type 1 (insulin-dependent) diabetic patients with nephropathy.

We investigated whether the glomerular synthesis of prostaglandins modulates the glomerular filtration rate and albuminuria in diabetic nephropathy. The urinary excretion of immunoreactive prostaglandin E2 (253 pg/min) was significantly elevated in eight Type 1 (insulin-dependent) diabetic women with nephropathy as compared with nine normoalbuminuric Type 1 diabetic women (95 pg/min) and 11 non-diabetic women (132 pg/min), respectively (p less than 0.01). Glomerular filtration rate (single bolus 51Cr-EDTA technique) and albuminuria (radioimmunoassay) were measured twice within two weeks in the eight Type 1 diabetic women with nephropathy. All eight patients were on a diabetic diet without sodium restriction. The study was performed as a randomized double-blind trial, with the patients receiving either indomethacin (150 mg/day) or placebo for three days prior to the kidney function studies. Indomethacin treatment induced a significant reduction in urinary prostaglandin E2 excretion (73%, p less than 0.01), glomerular filtration rate diminished from 120 +/- 18 to 106 +/- 17 ml/min/1.73 m2 (p less than 0.05), albuminuria declined from 148 to 69 micrograms/min (median and range) (p less than 0.05) and fractional clearance of albumin diminished 42% (p less than 0.05). Blood glucose concentrations were comparable during the placebo and indomethacin treatment, 13.4 +/- 4 versus 14.2 +/- 3 mmol/l, respectively. Our results suggest that glomerular filtration rate in early diabetic nephropathy is dependent on the enhanced glomerular synthesis of vasodilating prostaglandins.

Exaggerated hypotension by N-formylmethionylleucyl-phenylalanine in indomethacin pretreated rats. Role of toxic oxygen.

In pentobarbital anaesthetized rats the intravenous administration of the oligopeptide N-formyl-Met-Leu-Phe (FMLP) is shown to produce a short-lasting hypotension. When the animals have been pretreated with indomethacin, FMLP induces a marked and sustained blood pressure fall. This exaggerated hypotensive response to FMLP is absent in rats which have received a low dose of endotoxin at the day before, and is greatly reduced in animals treated with ascorbic acid or dimethylsulfoxide. In addition, the duration of hypotension is shortened in rats which have been partially depleted of leukocytes by daily methotrexate dosages, or which simultaneously receive drug treatments known to enhance vessel wall cyclic AMP levels like isoprenaline, glucagon or theophylline. The results suggest that after the FMLP administration toxic oxygen species generated by leukocytes contribute to the development of the cardiovascular dysfunction. Endothelial cAMP is suggested to control the sensitivity of the cardiovascular system to these reactive species.

Atriopeptin III improves renal functions in a ureter-obstructed rat kidney model.

The renal hemodynamic and excretory effects of atriopeptin III were studied in normal rat kidneys and in kidneys made dysfunctional by the application and release of a 24 h unilateral ureteral obstruction (UO24h), which decreased baseline glomerular filtration rate (GFR) by 80%. Atriopeptin III (0.5 nmol/kg i.v.) decreased blood pressure (10-15%) for more than 30 min, and increased urine flow rate and sodium excretion in normal and diseased kidneys for ca. 15 min. An initial enhancement of renal blood flow (ca. 20%) was apparent for less than 5 min. Atriopeptin III (bolus injection) temporarily enhanced the GFR 2-3-fold in the diseased (UO24h) kidneys, whereas no changes of GFR were noted in control kidneys. When atriopeptin III was continuously infused at a rate of 0.1 nmol/kg per min, GFR in UO24h kidneys increased from 0.28 +/- 0.08 ml/g per min to a stable level of 0.82 +/- 0.10 ml/g per min. Again, GFR in the control kidneys remained unaffected (1.25 +/- 0.08 ml/g per min). The enhancement of GFR in the UO24h kidney was associated with large increases of urine flow rate and sodium excretion.

Leukotriene production in rat peritoneal leukocytes requires intact energy metabolism.

Compounds which inhibit cellular production of ATP either by uncoupling of oxidative phosphorylation (valinomycin, carbonylcyanide-4-trifluoromethoxphenylhydrazone, and 2,4-dinitrophenol), glycolytic phosphorylation (2-deoxy-D-glucose) or by inhibiting respiratory-chain reactions (antimycin A) were all shown to inhibit calcium-ionophore A23187-induced leukotriene synthesis in rat peritoneal leukocytes at concentrations closely correlating with those needed to block ATP synthesis. In contrast, none of the compounds interfered with cyclo-oxygenase or other enzymes involved in arachidonate metabolism in these cells. Two well-known inhibitors of 5-lipoxygenase, nordihydroguaiaretic acid and phenidone, blocked LTB4 synthesis without affecting ATP production. In conclusion, rat peritoneal leukocyte leukotriene synthesis depends on intact energy metabolism.

Vascular effects in dogs of pinacidil (P 1134), a novel vasoactive antihypertensive agent.

In pentobarbital sodium-anaesthetized dogs, pinacidil was infused for approximately 5 min into the carotid, coronary, femoral or renal artery at a rate of 10 micrograms/kg per min. The infusion, which did not affect systemic blood pressure, rapidly and markedly increased blood flow to any of the regions studied. When given i.v., 0.2 mg/kg pinacidil caused a moderate reduction in mean arterial blood pressure (15-20 mmHg) associated with an increase in coronary and renal blood flow while femoral and carotid blood flow remained unchanged; 0.5 mg/kg led to a marked (40-60 mmHg) reduction in blood pressure associated with an increase in coronary blood flow whereas renal, carotid and femoral blood flow stabilized at control levels. Indomethacin (2.5 mg/kg i.v.) failed to reverse the hypotension induced by pinacidil. The results are in accord with the concept that the vascular effect of pinacidil is due to direct smooth muscle relaxation which does not depend on prostaglandin synthesis.

Clonidine decreases rat urine kallikrein excretion by alpha-adrenergic receptor stimulation.

In normal conscious female Sprague-Dawley rats, clonidine dose-dependently (10-300 micrograms/kg) decreased urine kallikrein excretion to approximately 40% of the control value. The effect of clonidine on urine enzyme excretion was inversely related to diuresis-natriuresis. Phenoxybenzamine (1 mg/kg) or phentolamine (10 mg/kg) antagonized the effect of clonidine on urine enzyme excretion. Consequently, rat urine kallikrein excretion might be reduced when alpha-adrenergic receptors are stimulated by clonidine.