Expression of calcitonin receptor-like receptor in human vascular tumours.

BACKGROUND: Vascular tumours such as Kaposi's sarcoma and capillary haemangioma are characterised by abnormal vascularisation and proliferation of endothelial cells or neoplastic cells. Adrenomedullin, a potent vasodilative peptide, and its receptor, calcitonin receptor-like receptor (CRLR), play an important part in angiogenesis. AIM: To establish whether this system also plays a part in vascular diseases, showing abnormal proliferation such as vascular tumours. METHODS: CRLR expression was investigated in several specimens of Kaposi's sarcoma and other vascular tumours, using immunohistochemical analysis with a previously described CRLR-specific polyclonal antibody and reverse transcriptase-polymerase chain reaction. RESULTS: Intense and specific CRLR-immunoreactive staining of neoplastic cells was observed in all specimens, which was of greater intensity than similar staining of adjacent normal endothelium. CONCLUSIONS: CRLR is expressed in vascular tumours and, with adrenomedullin, may have a role in neoplastic vascular growth.

Platelet-induced inhibition of adrenomedullin secretion.

Adrenomedullin is a biologically active peptide released from the vascular wall, which increases blood flow through its vasorelaxant effects and prevents platelet activation by stimulation of nitric oxide synthesis. The present study demonstrates that activated platelets suppress adrenomedullin secretion from vascular endothelial cells by releasing a factor that was identified as transforming growth factor (TGF)-beta1. Adrenomedullin levels were reduced by up to 40% and this effect was completely abrogated by the addition of latency-associated protein (LAP) or TGF-beta1-neutralizing antibody. Inhibition of adrenomedullin secretion in response to platelet aggregation may be an important mechanism in the induction of hemostasis.

Oxytocin in the male: an old hormone growing sexy with age.

It has long been a mystery of oxytocin research that males have similar levels of the hormone in their blood as females, but there is no known function associated with this. This review brings together some diverse literature to point out a possible role of oxytocin in the context of stress and sexuality.

Binding and internalization of leptin by porcine choroid plexus cells in culture.

Leptin is an adipocyte derived hormone with profound behavioural and metabolic effects exerted by both central and peripheral sites of action. One of its targets in the central nervous system appears to be the epithelial cells of the choroid plexus where leptin receptor (OB-R) expression is particularly high. The most abundant receptor subtype at this site is OB-Ra which is truncated at its intracellular part and has been suggested to serve functions such as leptin transport or clearance. The choroid plexus may thus be a site where receptor mediated exchange of leptin between cerebrospinal fluid and blood takes place. The study here shows that porcine plexus epithelia preserve their ability of OB-R expression when grown in culture. In addition, our experiments suggest that leptin is rapidly internalized upon binding to these cells supporting the view of an OB-R mediated transport of leptin across the choroid plexus.

Leptin receptor expression and suppressor of cytokine signaling transcript levels in high-fat-fed rats.

Several lines of evidence suggest that obese individuals have a higher set point for body weight regulation relative to lean subjects. Since obese rodents and humans have high serum levels of leptin, it has been hypothesized that this may be the result of an insensitivity to this weight reducing hormone. In this experiment we assessed whether feeding of a high-fat diet to rats affects leptin receptor (OB-R) transcript levels or induces up-regulation of the suppressors of leptin/cytokine induced signaling, SOCS-3 and PIAS-3. We found that despite a significant weight gain associated with markedly increased circulating leptin levels neither OB-R gene expression nor SOCS-3 or PIAS-3 mRNA levels were significantly altered in the high-fat fed rats. This was in contrast to control experiments where administration of exogenous leptin induced a several-fold increase in SOCS-3. It is concluded that high-caloric food intake per se is not sufficient to provoke suppression of leptin signaling via these factors in animals without genetic predisposition to obesity.

Bovine adrenals and hypothalamus are a major source of proscillaridin A- and ouabain-immunoreactivities.

Besides an isomer of the cardenolide ouabain, a material with a similar HPLC retention time as ouabain but cross-reactivating with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump is known to circulate in human blood plasma (B. SICH et al., Hypertension 27, 1073-1078 (1996).). The concentrations of both substances are known to correlate with the blood pressure. It was the intention of this work to localize tissues that contain the highest concentrations of the proscillaridin A immunoreactive material, to correlate its concentration with that of ouabain and to get information whether the concentration of this material simply reflects the number of sodium pumps of the tissue extracted. Specific antibodies for each cardiotonic steroid were used to test the tissue concentration. This report shows that in bovine tissues the distribution pattern of proscillaridin A and ouabain immunoreactivities are similar and that hypothalamus and adrenals show the highest concentrations. The cross-reactive material did not reflect the number of sodium pumps per g of wet weight tissue as measured by [3H]ouabain binding. Therefore, it is unlikely that the tissue concentrations in both immunoreactivities reflects the tissue capacity of sodium pumps labeled with cardiotonic steroids via the blood plasma. The study rather favors the concept that two different types of inhibitors of the sodium pump exist within both tissues.

High levels of circulating adrenomedullin in severe illness: correlation with C-reactive protein and evidence against the adrenal medulla as site of origin.

Adrenomedullin (AM) is a novel vasorelaxing peptide which was originally isolated from the extracts of human pheochromocytoma. It is produced by a number of organs among which the adrenal gland exhibits by far the highest concentrations. The peptide circulates in blood and its plasma levels have been reported to be increased in several diseases such as renal failure and sepsis. In the present study plasma concentrations of AM were measured in various forms of severe illness and compared to clinical and biochemical parameters in order to gain an insight into the factors controlling the plasma levels of this peptide. The highest concentrations of AM were found in patients with sepsis (344.4 +/- 60.4 pg/ml, n = 16) who exhibited up to 12-fold higher levels than a group of healthy subjects (74.1 +/- 4.1 pg/ml, n = 20). Markedly elevated levels were also measured in hemorrhagic (250.1 +/- 37.9 pg/ml, n = 9) and cardiogenic (216.2 +/- 29.4 pg/ml, n = 7) shock as well as in patients with cancer of the gastrointestinal tract (155.6 +/- 32.5 pg/ml, n = 11) or the lungs (146.5 +/- 19.1 pg/ml, n = 22). Plasma AM levels were positively correlated with serum creatinine concentrations in shock (r = 0.06, p < 0.001) and with C-reactive protein levels in patients with cancer (r = 0.64, p < 0.001) or sepsis (r = 0.63, p < 0.01). In order to examine the potential role of the adrenal gland as a site of AM release, hypoglycemia was induced in a group of healthy volunteers by graded infusion of insulin. Despite a more than 20-fold increase in plasma adrenalin indicating maximal stimulation of the adrenal medulla, no significant alterations of the plasma AM levels were observed. The study demonstrates that not only sepsis but also various forms of cancer and shock are associated with high levels of circulating AM. The correlation with C-reactive protein levels suggests a role of cytokines in mediating the elevations in plasma AM observed in sepsis and cancer. Reduced clearance of the peptide by the kidneys may be one of the mechanisms involved in the accumulation of AM in shock. The adrenal gland appears not to be a major source for circulating AM.

Leptin: a potent inhibitor of insulin secretion.

The hormone leptin is expressed and secreted by the adipose tissue and impacts on the central nervous system. Leptin is involved in the regulation of energy balance, satiety, and body composition. The lack of active leptin results in obesity, high food intake, hyperglycemia, and hyperinsulinemia. We present data supporting effects of leptin on the endocrine pancreas. We found the leptin receptor to be expressed in insulin- and glucagon-secretin cells derived from mouse, hamster, and rat pancreas. In the isolated perfused rat pancreas leptin is a potent inhibitor of basal and glucose-induced insulin secretion, especially during the first phase of the insulin response. At isolated mouse islets and insulin-secreting INS-1 cells leptin reduced promptly and persistently the intracellular Ca2+ levels. Cytoplasmic Ca2+ oscillation amplitude was decreased and the oscillation frequency increased. These findings suggest functional active receptors for leptin on insulin-secreting B-cells. Therefore, leptin is a metabolic hormone and not only a signal to the brain indicating filled fat stores. Our data suggest that leptin is also a signal back to the endocrine pancreas that no more insulin is required to replenish fat stores. Thus, an "adipo-insular axis" operating with two arms exists: insulin and glucagon are signals to the adipocyte. This releases leptin, which could be the mediator of the respective feedback to the pancreas. A defective leptin suppression of insulin secretion could contribute to hyperinsulinemia and disturbances of glucose metabolism.

Characterization of ouabain-like immunoreactivity in human urine.

OBJECTIVE: To characterize ouabain-like immunoreactivity in human urine. METHODS: Sensitive radioimmunoassay for ouabain characterized by high-performance liquid chromatography. RESULTS: Serial dilution of urinary immunoreactive ouabain paralleled the standard curve, but not so plasma immunoreactive ouabain. Intravenous administration of 86 nmol (62.5 micrograms) ouabain caused a rapid rise in ouabain immunoreactivity in plasma of healthy volunteers with a maximum of 1.7 nmol/l 8 min after injection and returned to basal levels after 6 h. Ouabain immunoreactivity rose to 36 nmol/l in urine, suggesting that exogenously administered ouabain can be measured reliably in plasma and urine. Analytical reversed-phase high-performance liquid chromatography (isopropanol-propanol biphasic gradient; linear acetonitrile gradient) of sample extracts before assay demonstrated measurable amounts of ouabain-related material only in native urine, but not in plasma. When plasma and urine were spiked with ouabain standard or normal volunteers were injected with ouabain, the assay reliably measured ouabain. CONCLUSION: A substance closely related to ouabain can be detected in urine, but circulates, if at all, in small amounts in human plasma.

Radiommunological measurement of leptin in plasma of obese and diabetic human subjects.

Studies in mice have identified the ob gene product, leptin, as a signaling factor regulating body weight homeostasis and energy balance. Defective production of the encoded protein may be one of the causes for the development of obesity. Using a high affinity antibody, that in immunohistochemical studies specifically stained human adipocytes, a radioimmunoassay was established and leptin immunoreactivity was quantified in plasma of lean and obese human subjects. Chromatographic analysis suggested that the immunoreactive material in plasma is identical to that found in extracts from human fat and represent a protein with a molecular size of approximately 16 kD. Fasting levels were measured in plasma of 75 lean and obese human subjects (body mass index (BMI) 17.7 - 87.3). The mean concentration of leptin in plasma of lean subjects (BMI < or = 28) was 69.3 +/- 36.9 fmol/ml plasma (mean +/- SD, n=27). The highest concentration measured in obese was 533.3 fmol/ml plasma. The levels showed a strong positive correlation with BMI (r=0.77, p<0.001). A subgroup of diabetic patients did not significantly differ in their leptin plasma levels from non-diabetic subjects with similar BMI.

Gene expression of natriuretic peptide receptors in myocardial cells.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that serve to unload the heart through their effects on the kidney and vasculature. Whether the heart itself represents a site of action for these peptides is currently the subject of debate. Although functional studies indicate that ANP has some effects on isolated myocytes, several studies have been unable to detect binding of the hormone to these cells. The present study demonstrates that the genes for all three natriuretic peptide receptor (NPR) subtypes, NPR-A, NPR-B, and NPR-C, are expressed in the rat heart. For microlocalization of the receptor mRNAs in myocytes and nonmyocytic cells, a combination of cell isolation and reverse transcription-polymerase chain reaction (RT-PCR) was used. Cardiac myocytes were isolated by enzymatic dissociation of rat ventricular tissue, purified by density gradient centrifugation, and collected as single cells under microscopic control. Analysis by RT-PCR revealed the presence of transcripts for NPR-A as well as NPR-B and NPR-C. However, cGMP generation in purified myocytes was stimulated only by ANP and BNP, which specifically bind to NPR-A, whereas C-type natriuretic peptide (CNP, an NPR-B agonist) was ineffective. Therefore, rat ventricular myocytes appear to produce predominantly NPR-A. The expression of NPR-B may be low or even absent. The mRNAs for all three NPRs were also found in cultures of fibroblasts from the rat heart. In contrast to the myocytes, large increases in cGMP were observed in response not only to ANP but also to CNP.

Gene expression of atrial natriuretic peptide in rat papillary muscle. Rapid induction by mechanical loading.

The effect of mechanical stretch on protein synthesis and the expression of the gene for atrial natriuretic peptide (ANP) was examined in electrically paced, isolated papillary muscles from rat heart. Incorporation of [3H]phenylalanine into protein increased only in stretched but not in unloaded muscles. Five hours of stretching increased ANP mRNA levels more than threefold as compared to freshly excised papillary muscles. A drastic fall in ANP mRNA levels was observed in unloaded muscles over this time. These data indicate that papillary muscles similar to other ventricular tissue are capable of activating ANP gene expression in response to increased load. The effect occurs in vitro and does not depend on circulating or nervous factors. The unexpected rapid induction of ANP gene expression in such a particular structure of the heart raises the possibility of local actions of ventricular ANP.

Elevated plasma concentration of neuropeptide Y in adolescents with primary hypertension.

Neuropeptide Y (NPY) has been recently characterised as a circulating vasoconstrictor peptide which is co-stored with noradrenaline (NA) in sympathetic neurons. We measured NPY by radioimmunoassay and NA by HPLC in plasma of ten healthy volunteers (23-27 years of age) during bicycle ergometry and found a rapid increase of both NPY and NA during exercise. NPY rose from 1.3 +/- 0.5 to 9.6 +/- 7.8 pmol/l and NA from 1.3 +/- 0.3 to 10.8 +/- 5.6 mnol/l (mean +/- SD). Following maximal exercise NA disappeared more rapidly from plasma than NPY. Compared with these healthy volunteers, plasma NPY was found to be elevated in 23 children and adolescents aged 9-18 years with borderline primary hypertension (NPY 3.1 +/- 1.7 pmol/l, P < 0.01). Basal NPY was also elevated when compared with 21 age-matched pediatric controls (P < 0.05). The bicycle ergometry protocol performed in 23 patients separated ten adolescents with normal basal and exercise blood pressure from 13 with high BP also during ergometry. In the latter group, NPY rose to 11.9 +/- 7.3 pmol/l and NA to 12.3 +/- 8.6 nmol/l during exercise. Treatment of the hypertensive patients with the beta-adrenergic blocker atenolol (50 mg per day) lowered basal and exercise BP. Heart rate fell during atenolol treatment from 92 +/- 19 to 72 +/- 15 beats/min. Treatment did not alter plasma concentrations under basal conditions and during exercise (NPY from 2.8 +/- 2.1 to 11.7 +/- 5.3 pmol/l and NA from 2.0 +/- 0.8 to 15.6 +/- 14.1 nmol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of catecholamine depletion and denervation on neuropeptide Y(NPY) and tyrosine-hydroxylase (TH) mRNA levels in rat sympathetic ganglia.

Neuropeptide Y (NPY) and noradrenaline (NA) are synthesized and stored in sympathetic nerves and concomitantly released in response to appropriate stimuli. The two substances have been reported to interact on various levels: postjunctionally, by mutually potentiating their vasoconstrictor effects, prejunctionally, by inhibiting each other's release. The possibility of an interaction on the levels of their synthesis was investigated in this study. Specific cDNA probes were used for the measurement of the steady state levels of the mRNAs encoding prepro-NPY and tyrosine hydroxylase (TH) in the superior sympathetic cervical and stellate ganglia of rats. Reserpine (5 mg/kg) was administered for inducing catecholamine depletion. This caused a large decrease in the NA content of the heart associated with an about 50% reduction in cardiac NPY levels. Ganglionic NPY and TH mRNA levels increased 3-6 fold as compared to vehicle treated animals. To determine whether this effect was due to transynaptic induction, superior cervical ganglia were decentralized in a subgroup of rats. Decentralized ganglia displayed significantly lower NPY and TH mRNA levels than intact ones. The response to reserpine was almost completely prevented by decentralization. These Observations indicate that the activation of gene expression of NPY and TH by reserpine depends on intact ganglionic innervation and is therefore reflexly mediated. Trans-synaptic induction may regulate NPY and TH mRNA levels also under basal conditions.

Atrial natriuretic peptide (ANP) responsiveness in patients with hypothyroidism.

Hypothyroidism is known to be associated with abnormalities of kidney function; recently, low atrial natriuretic peptide (ANP) plasma levels have been reported. Aim of the study was to asses ANP, sodium and water responsiveness to an acute saline load. Twelve patients with established primary hypothyroidism and 9 control subjects were studied. ANP was determined in plasma by RIA with extraction, prior to and after the infusion of saline, 500 ml/h for 4 hours. On a similar albeit liberal sodium diet hypothyroid patients excreted less sodium and water (74 +/- 33 (SD) mumol/min and 0.69 +/- 0.15 ml/min, respectively) than control subjects (110 +/- 52 mumol/min; P < 0.05 and 1.06 +/- 0.53 ml/min; P < 0.025, respectively). However, the infusion of saline resulted in a 3-fold increase of sodium output and more than 2-fold increase in urine flow. The exaggerated responsiveness in sodium excretion in patients with hypothyroidism was associated with significantly decreased pre-infusion ANP plasma levels (16.1 +/- 11.1 pg/ml vs. 44.4 +/- 14.4 pg/ml; P < 0.001) and also with sluggish response to the volume expansion (+24% vs. +48%). A significant correlation was found between serum T4 levels and plasma ANP concentrations in 8 patients (r = 0.689; P < 0.05). Although hypothyroid patients tend to retain sodium on a liberal salt diet, their kidney is capable of vigorously eliminating excess sodium when challenged with an acute saline load. This exaggerated responsiveness of sodium excretion can be demonstrated in spite of a sluggish response in ANP. Subnormal ANP levels in hypothyroidism are probably the result of thyroid deficiency.

Serial measurements of neuropeptide Y in plasma for monitoring neuroblastoma in children.

Neuropeptide Y (NPY) was studied as a marker for neuroblastoma in 12 children. All but one patient with neuroblastoma had elevated plasma NPY concentrations at diagnosis. During treatment NPY values returned to normal in 9 of 12 children. All three children without normalization of plasma NPY values died; two of them had a relapse and the third died of toxic effects. Plasma NPY appears to be a sensitive marker of neuroblastoma.

Identification of neuropeptide Y receptors in cultured astrocytes from neonatal rat brain.

Specific binding sites for neuropeptide Y could be demonstrated in primary cultures of astrocytes from neonatal rat brain. Neuropeptide Y binding was saturable, reversible, and temperature dependent as revealed by saturation studies and kinetic experiments. Scatchard analysis of equilibrium binding data indicated a single population of high-affinity binding sites with respective KD and Bmax values of 0.43 nM and 6.9 fmol/2.7 x 10(5) cells. Physiological responses induced by neuropeptide Y could be detected in a distinct subpopulation of cultured astrocytes on the basis of two criteria: 1) electrophysiological responses and 2) single cell measurements of changes in [Ca2+]i. In that fraction of cells responding (20-70%, varying among cultures from different preparations), brief application of neuropeptide Y led to a membrane potential depolarization, lasting several minutes. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, neuropeptide Y induced an inward current with a similar time course as the neuropeptide Y-induced membrane depolarization. As detected by single cell microfluorimetric (fura-2) measurements neuropeptide Y induced an increase of [Ca2+]i which was caused by the entry of extracellular Ca2+. Both the [Ca2+]i increase and the electrophysiological responses were unaffected by pretreatment of the astrocytes with pertussis toxin.

Tyrosine-hydroxylase-containing vagal afferent neurons in the rat nodose ganglion are independent from neuropeptide-Y-containing populations and project to esophagus and stomach.

Immunoreactivity to the rate limiting enzyme of catecholamine synthesis, tyrosine hydroxylase, has been described in the inferior sensory (= nodose) ganglion of the vagal nerve in the rat. The aim of the present study was to characterize further this neuronal population. The neurons do not represent displaced autonomic efferent neurons, since they do not receive synaptic input, as indicated by the absence of synaptophysin-immunoreactive terminals. In addition to the immunoreactivity to tyrosine hydroxylase, a tyrosine hydroxylase cRNA probe hybridizes with nodose ganglion neurons as demonstrated by in situ hybridization and Northern blotting. Many but not all of the tyrosine hydroxylase-immunoreactive neurons are also immunoreactive to the dopamine synthesizing enzyme, aromatic-L-amino-acid-decarboxylase, but lack the noradrenaline-synthesizing enzyme, dopamine-beta-hydroxylase, thus favoring synthesis of dopamine. Neuropeptide Y, which is often colocalized with catecholamines, is also present in a subset of nodose ganglion neurons, as indicated by immunohistochemistry, in situ hybridization and Northern blotting. However, double-labeling immunofluorescence has revealed that these two antigens are localized in different cell populations. Retrograde neuronal tracing utilizing fluorescent dyes (Fast blue, Fluoro-gold) combined with tyrosine hydroxylase immunohistochemistry has demonstrated that the esophagus and stomach are peripheral targets of tyrosine-hydroxylase-containing vagal viscero-afferent neurons.

Diuresis and natriuresis following isotonic saline infusion in healthy young volunteers before, during, and after HDT.

In the present study the response to acute saline loading was investigated. During a 24-day study period six male subjects followed a standardized diet including a daily intake of 40 ml water and 125 mg NaCl per kg body weight. Before, during, and after a ten-day period of 6 degrees head down tilt (HDT) each volunteer received an intravenous 0.9% saline infusion of 22 ml/kg body weight over 20 minutes. HDT produced significant losses in body weight and in blood volume, but the responses to saline loading were similar during all phases of the study. Plasma levels of atrial natriuretic peptide (ANP) did not increase, while plasma levels of cyclic GMP increased by about 40% 90 minutes after each infusion. Urine flow nearly doubled during second hour post-infusion. Sodium excretion showed a 3-fold increase and remained elevated during the third hour, while potassium excretion was significantly reduced. Urinary excretion of cyclic GMP reached a peak during the second hour post-infusion. At the end of these short-term periods the cumulative water- and sodium-balance data disclosed that only about 20% of the infused water and less than 15% of the infused sodium was excreted during each experiment. In addition to the short-term renal response, urine flow and sodium excretion remained significantly elevated for more than 48 hours after each saline load. The long-term renal response was paralleled by an increased excretion of urinary cyclic GMP. HDT produced significant changes in body fluid distribution, but only minor changes in the regulatory responses to an acute saline load. We conclude from these data that the excretion of an acute isotonic saline load requires several days and that the renal response appears to be independent of the secretion of ANP from the heart.

Effects of endothelin on the renal microcirculation of the split hydronephrotic rat kidney.

We have examined the effects of endothelin (ET) on the renal microcirculation by in vivo microscopy using the model of the split hydronephrotic rat kidney. ET, a potent vasoconstrictor peptide synthesized by vascular endothelial cells, showed marked and long-lasting effects on glomerular blood flow and vessel diameters in various segments of the renal vascular bed. Intravenously applied ET (100 ng/min/kg) increased systemic blood pressure from 123 +/- 7 to 156 +/- 4 mm Hg, decreased glomerular blood flow by 70%, and preferentially constricted larger preglomerular vessels, e.g. the arcuate artery. The competitive leukotriene antagonist FPL55712 significantly attenuated the vasoconstrictor response of the larger vessels. Local ET administration decreased glomerular blood flow in a dose-dependent manner (50% reduction at a concentration of 2.6 +/- 0.7 x 10(-9) M) and constricted smaller vessel segments, e.g. the afferent and efferent arterioles near the glomerulus. The constriction induced by ET was not significantly affected by the Ca2+ channel blocker nitrendipine (2.8 x 10(-6) to 1.1 x 10(-5) M). We conclude that intravenous ET effects are probably mediated by leukotrienes, inducing constriction of larger renal vessels. Locally administered ET acts directly on the renal vasculature, especially on smaller vessels.