Anette Hemsén

Radioimmunoassay for neuropeptide Y (NPY) : chromatographic characterization of immunoreactivity in plasma and tissue extracts

A sensitive and specific radioimmunoassay was developed to determine the occurrence and concentration of neuropeptide Y (NPY) in plasma and tissue extracts. Furthermore, NPY-like immunoreactivity (NPY-Li) was characterized by means of three different chromatographic systems. The NPY antiserum used (NI) did not cross-react with related peptides of the pancreatic polypeptide family except avian pancreatic polypeptide (1% cross-reactivity). Unextracted plasma contained high molecular weight proteins which interfered in the assay. Acid ethanol extraction removed this protein interference allowing a 90% recovery of NPY-Li. The content of NPY-Li in human plasma from healthy subjects was close to or below the detection limit (less than 22 pmol/l). Sympathetic nerve stimulation in the cat increased the output of NPY-Li from the splenic vein suggesting the release of this peptide upon sympathetic activation. The major peak of NPY-Li in spleen extracts and splenic vein plasma co-eluted with synthetic porcine NPY and a minor peak with larger Stokes radius was also present. The present radioimmunoassay enables further studies on the physiological and pathophysiological role of NPY.

Co-release of neuropeptide Y and catecholamines during physical exercise in man

Venous plasma levels of neuropeptide Y-like immunoreactivity (with chromatographic properties of synthetic neuropeptide Y) increased in parallel with catecholamines, heart rate and blood pressure during graded physical exercise in man. The plasma levels of neuropeptide Y correlated better with the levels of noradrenaline than adrenaline, suggesting release of a neural origin. Taken together with previous results, this suggests that neuropeptide Y is released together with noradrenaline upon sympathetic activation during physiological conditions in man. Determinations of plasma neuropeptide Y may therefore be valuable in the assessment of sympathetic nerve activity.

Neuropeptide Y-like immunoreactivity in adrenaline cells of adrenal medulla and in tumors and plasma of pheochromocytoma patients

The occurrence of neuropeptide Y (NPY)-like immunoreactivity (LI) in the adrenal gland of several species as well as in tumor tissue and plasma from pheochromocytoma patients was investigated. NPY-LI was present in chromaffin cells of the adrenaline type in all species investigated except in the pig, as demonstrated by a colocalization of NPY-LI and the adrenaline-synthetizing enzyme phenylethanolamine N-methyltransferase (PNMT). NPY-LI in the adrenaline cells of the cat was clearly separated from the neurotensin-LI in the noradrenaline dopamine-beta-hydroxylase-positive, PNMT-negative cells. NPY-LI seems to co-exist with enkephalin-like material in the chromaffin cells. In addition, NPY-LI was present in nerves both within the adrenal cortex and medulla. The highest levels of NPY-LI were found in mouse and cat, while only a very low amount of NPY-LI was present in the pig adrenal. Characterization of the adrenal NPY-LI by reversed-phase high-performance liquid chromatography revealed that the main peak was similar to porcine NPY. In addition, two minor peaks of NPY-LI were present. High levels of NPY-LI were found in plasma and tumors from the pheochromocytoma patients. During manipulation of the tumors upon surgical removal, there was a marked increase in plasma NPY-LI in parallel with the raise in catecholamines and in blood pressure. At least two forms of NPY-LI were present in plasma and tumor extracts from pheochromocytoma patients with the main peak corresponding to porcine NPY. Since NPY exerts vasoconstrictor effects, it may be postulated that NPY contributes to the adrenal cardiovascular response and to the hypertension seen in pheochromocytoma patients.

Neuropeptide Y receptor in pig spleen: binding characteristics, reduction of cyclic AMP formation and calcium antagonist inhibition of vasoconstriction.

Specific, high-affinity binding sites for 125I-porcine neuropeptide Y (NPY) were demonstrated in membranes from the pig spleen. The equilibrium dissociation constant (KD) of the receptor 125I-NPY complex was 532 +/- 87 pM and the maximal number of specific binding sites (Bmax) 23 +/- 3 fmol/mg protein. The Scatchard plot for 125I-NPY binding under equilibrium conditions showed a best-fit to a straight line, whereas the dissociation appeared biphasic. 125I-NPY binding was unaffected by adrenoceptor antagonists and was inhibited by the guanosine triphosphate (GTP) analogue guanylylimidodiphosphate, suggesting regulation by a GTP binding protein. A series of NPY analogues showed a good correlation between binding, inhibition of forskolin-induced cyclic adenosine monophosphate (cAMP) formation and vasoconstrictor activity in vivo. A large carboxyl terminal portion of NPY and the carboxyl terminal amide were essential for binding, inhibition of cAMP formation and vasoconstrictor effects. The NPY fragment 13-36, which has been reported to act only on prejunctional NPY receptors, showed only a 10-fold lower potency than NPY-(1-36) both in binding to splenic membranes and vasoconstrictor activity in vivo. Phenylephrine increased phosphatidyl inositol turnover whereas NPY-(1-36) or -(13-36) did not induce formation of inositol phosphates. The calcium antagonists felodipine and nifedipine attenuated the splenic vasoconstrictor response to NPY in vivo but not the NPY-evoked inhibition of cAMP accumulation or the specific binding of 125I-NPY.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue specific distribution, clearance and vascular effects of endothelin in the pig

Endothelin-like immunoreactivity (-LI) was detected in the kidney, spleen, skeletal muscle, lung and in plasma of the pig. The highest tissue levels were found in the lung (1.5 +/- 0.07 pmol/g) and the lowest in the muscle (0.08 +/- 0.02 pmol/g). I.v. infusion of endothelin (20 pmol/kg/min for 20 min) increased plasma endothelin-LI from 18 +/- 3 to 628 +/- 109 pM and renal, splenic and femoral vascular resistance by 570, 430 and 50%, respectively. There was a close correlation between the increase in plasma endothelin-LI and the vasoconstrictor effects. During the infusion 92, 82 and 76% of arterial endothelin-LI was removed by the kidney, spleen and hindlimb, respectively, whereas there was no clearance over the lung or degradation in plasma. After the infusion, arterial endothelin-LI decreased with a half life of 77 s, while the renal and splenic vasoconstriction persisted for 60 min. Characterization by reversed phase HPLC revealed that endothelin-LI in plasma and tissue co-eluted with synthetic endothelin. It is concluded that endothelin has tissue specific distribution, clearance and vascular effects in the pig.

Metabolism of Big endothelin-1 (1–38) and (22–38) in the human circulation in relation to production of endothelin-1 (1–21)

Healthy male volunteers received intravenous infusions of Big endothelin (ET)-1 (1-38) or Big ET-1 (22-38). Blood samples were drawn from catheters in the brachial and pulmonary arteries and the hepatic, renal, jugular and deep forearm veins. The in vivo half-lives of circulating plasma Big ET-1 (1-38) were 6.6 +/- 0.3 min for the initial phase and 23 +/- 1.4 min for the late phase. The corresponding half-lives of Big ET-1 (22-38) were considerably shorter, being 0.9 +/- 0.03 min (P < 0.01) and 3.1 +/- 0.4 min (P < 0.01), respectively. This was concordant with the efficient regional clearance of Big ET-1 (22-38), which was most prominent in the forearm muscle (51 +/- 3%), liver (44 +/- 5%) and kidney (43 +/- 3%) and less pronounced in the lungs (14 +/- 2%) and brain (22 +/- 5%). Significant fractional extraction of Big ET-1 (1-38) was only found for the liver (30 +/- 2%) and kidney (44 +/- 3%). During the infusion of Big ET-1 (1-38) a positive veno-arterial gradient of ET-1-LI was noted only for the kidney, indicating production of ET-1. In conclusion, whereas Big ET-1 (22-38) is eliminated in skeletal muscle, splanchnic, renal, pulmonary and cerebral vascular beds, Big ET-1 (1-38) is extracted mainly in the renal and splanchnic vasculature. Furthermore, plasma half-life of Big ET-1 (1-38) is much longer than that of both ET-1 and Big ET-1 (22-38) in man. Thus, for investigation of the secretory activity of the ET-1-system measurements of Big ET-1 (1-38) levels may be a better approach.

Pharmacology of noradrenaline and neuropeptide tyrosine (NPY)‐mediated sympathetic cotransmission

Summary— Pharmacological and physiological aspects for neuropeptide Y (NPY) and noradrenaline (NA) cotransmission have been studied in the peripheral sympathetic nervous control of blood vessels, heart, spleen and vas deferens. NPY coexists with NA in large dense cored vesicles and is released compared to NA mainly upon high frequency stimulation or strong reflex sympathetic activation. NPY release is inhibited via prejunctional α‐2 adrenoceptors and adenosine receptors but facilitated by angiotensin II or β‐receptor activation. NPY exerts prejunctional inhibitory actions on both NA and NPY release, enhances the vasoconstrictor effect of NA and evokes potent, long‐lasting vasoconstriction. Specific receptor mechanisms for NPY exist at both the pre‐ and postjunctional levels; a large amidated C‐terminal portion of NPY is necessary for receptor binding, inhibition of cyclic AMP formation and vasoconstrictor effects. Denervation results in supersensitivity for both NA and NPY‐evoked vasoconstriction. Reserpine pretreatment is associated with depletion of NA as well as NPY; the effect on NPY is entirely dependent on an intact nerve activity. Reserpine treatment combined with preganglionic denervation depletes NA by 99% while NPY levels are maintained intact. The characteristic appearance of the nerve stimulation evoked vasoconstrictor response with a high correlation to NPY outflow after reserpine treatment, suggests that NPY may be involved as a transmitter in a variety of vascular beds. NPY‐synthesis in ganglia seems to be regulated by nicotinic receptor activity; secondary stimulation by eg reserpine stimulates and nicotine antagonists decrease NPY‐synthesis. Many classical pharmacological agents including guanethidine, clonidine, yohimbine, angiotensin II, nicotine and desipramine influence NPY release. A complex interplay therefore seems to occur at both the pre‐ and postjunctional levels of transmission for the classical transmitter NA and the coexisting peptide NPY, creating a great diversity of chemical signalling potential.

Sample handling techniques when analyzing regulatory peptides

Collection of blood samples in prechilled heparinized tubes, rapid cooling and centrifugation at 4 degrees C were found to be more important than the enzyme inhibitors aprotinin and EDTA in preserving immunoreactive neuropeptide Y. Nine months after storage of plasma in the frozen state at -20 degrees C or -80 degrees C the recovery of NPY was about 50% of the recovery at immediate analysis. Synthetic substance P added to guinea pig plasma at 37 degrees C disappeared almost entirely within 30 seconds as measured by radioimmunoassay while the concentrations of neurokinin A and neuropeptide K decreased only to a minor extent during a 20 min observation period. The total concentration of immunoreactive substance P and neurokinin A in boiled aqueous and acetic acid extracts of rat dorsal spinal cord was on the other hand stable for 72 h at 4 degrees C, 24 h at room temperature and after freezing and thawing three times. However, chromatographic analysis indicated that the immunoreactivity became increasingly more heterogenous in the samples particularily at room temperature. Acid ethanol and Sep Pak extraction of plasma samples resulted in almost 90% recovery of neuropeptide Y, neuropeptide K and calcitonin gene-related peptide while removing crossreacting substances with high molecular weight.

Occurrence, specific binding sites and functional effects of endothelin in human cardiopulmonary tissue.

Endothelin (ET)-like immunoreactivity (-LI) was detected in the human cardiopulmonary system, with the highest levels being found in the left anterior descending coronary artery, followed by the lung, right atrium, pulmonary artery, bronchus, pulmonary vein and left ventricle. Chromatographic characterization showed that the ET-LI in the lung and left ventricle corresponded to synthetic ET-1. Specific, high-affinity binding sites for ET-1, with an extremely slow dissociation rate, were found in the lung, right atrium and left ventricle. Displacement studies revealed a rank order of potency of ET-1 greater than ET-2 and sarafotoxin 6b greater than ET-3 and big ET-1. Scatchard analysis indicated a single receptor population in the lung (KD 1.53 x 10(-10) M) and left ventricle (KD 3.0 x 10(-11) M). In functional experiments, ET-1 evoked concentration-dependent, long-lasting vasoconstriction of a higher potency than that evoked by ET-2 and ET-3 in epicardial coronary arteries as well as in pulmonary arteries. ET-1 and ET-2 also showed bronchoconstrictor activity at considerably lower concentrations (threshold 10(-11) M) of ET-1 than those needed to cause vasoconstriction (10(-9) M). ET-LI, mainly consisting of ET-1, occurs in human cardiopulmonary tissue. Specific, high-affinity sites with irreversible binding for ET-1 are found in both the heart and lung. ET-1 is more potent than ET-2 or ET-3 in displacing ET-1 binding and in causing vasoconstriction and bronchoconstriction. Thus, in the human heart and lung, ET-1 seems to be the most abundant and biologically active of the endothelin peptides.

Neuropeptide Y and catecholamine synthesizing enzymes and their mRNAs in rat sympathetic neurons and adrenal glands: Studies on expression, synthesis and axonal transport after pharmacological and experimental manipulations using hybridization techniques and radioimmunoassay

The effects of reserpine treatment (10 mg/kg, i.p.) on the content of neuropeptide Y-like immunoreactivity and catecholamines were compared with the levels of mRNA coding for neuropeptide Y, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rat sympathetic neurons and adrenal gland. A reversible depletion of neuropeptide Y-like immunoreactivity was observed in the right atrium of the heart, kidney and masseter muscle, while the immunoreactive neuropeptide Y content in the stellate and lumbar sympathetic ganglia and its axonal transport in the sciatic nerve increased following reserpine. The increase in the stellate ganglion was maximal at 48 h and absent 9 days after reserpine treatment. The expression of neuropeptide Y mRNA and tyrosine hydroxylase mRNA in both the stellate and the superior cervical ganglion increased earlier than the neuropeptide Y content, with a clear cut two-fold elevation at 24 h after reserpine. The increase in both mRNAs in the superior cervical ganglion and the depletion of neuropeptide Y, but not of noradrenaline, in terminal areas was prevented after pretreatment both with a nicotinic receptor antagonist (chlorisondamine) and with surgical preganglionic denervation. A marked (75-90%) depletion of neuropeptide Y-like immunoreactivity and adrenaline in the adrenal gland, concomitant with 3-4-fold increases in neuropeptide Y mRNA and tyrosine hydroxylase mRNA expression, was present at 24 h after reserpine treatment. Also in the adrenal gland, there was a reversal of the reserpine-induced increase in neuropeptide Y mRNA and tyrosine hydroxylase mRNA and depletion of neuropeptide Y and adrenaline following splanchnic denervation. Pharmacological, ganglionic blockade prevented the depletion of neuropeptide Y and the increased expression of neuropeptide Y mRNA, but not fully, the tyrosine hydroxylase mRNA elevation. In addition, a marked decrease in phenylethanolamine N-methyltransferase mRNA levels was noted after reserpine. This decrease was reversed by denervation and by ganglionic blockade. Denervation alone led to a small but significant decrease in all mRNAs examined both in the superior cervical ganglion and the adrenal medulla. The present data suggest that the depletion of neuropeptide Y-like immunoreactivity in sympathetic nerves and in the adrenal gland after reserpine is associated with a compensatory increase in neuropeptide Y synthesis and axonal transport, most likely due to increased nicotinic receptor stimulation. Whereas the reserpine depletion of neuropeptide Y in both sympathetic nerves and adrenal gland is related to neuronal activation, adrenal but not nerve terminal depletion of catecholamines can be prevented by the ganglionic blocker chlorisondamine.4+e difference in effect of pharmacological ganglionic