Pasireotide (SOM230) protects the retina in animal models of ischemia induced retinopathies.

The neuropeptide somatostatin and selective analogs for the sst(2/5) receptor subtypes provided neuroprotection against retinal chemical ischemia ex vivo and AMPA [(RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide] induced retinal toxicity in vivo, when employed in micromolar concentrations (Mastrodimou et al., 2005; Kiagiadaki and Thermos, 2008). The aim of the present study was to investigate the neuroprotective properties of a new metabolically stable agent pasireotide (SOM230) in the above mentioned retinal models of ischemia. Adult Sprague Dawley (250-350 g) rats were employed. For the ex vivo experiments, retinal eye cups were incubated with PBS or the chemical ischemia mixture [iodoacetic acid (5 mM)/sodium cyanide (25 mM)] in the absence or presence of SOM230 (10(-7)-10(-5) M) alone or in the presence of the sst(2) antagonist CYN-154806 (10(-7) or 10(-5) M). In the in vivo model, the animals received intravitreally: PBS (50 mM), AMPA (42 nmol/eye) or AMPA (42 nmol) in combination with SOM230 (10(-7)-10(-5) M). Immunohistochemistry studies using antisera against bNOS, a marker for brain/neuronal NOS containing amacrine cells, protein kinase C (PKC) a marker for rod bipolar cells, and TUNEL studies in conjunction with FACS analysis were employed to examine retinal cell loss and protection. Chemical ischemia led to a loss of bNOS and PKC immunoreactivity which was reversed by SOM230. Partial and full protection of bNOS and PKC immunoreactive neurons, respectively, was observed even at the low concentration of 10(-7) M. The neuroprotective actions of SOM230 (10(-7) or 10(-5) M) were reversed by CYN-154806 (10(-7) or 10(-5) M, respectively). Similarly, SOM230 (10(-7), 10(-6), 10(-5) M) provided neuroprotection in the in vivo model. The dose of 10(-7) M prevented the loss of the bNOS cells and provided almost full protection. These data were substantiated by TUNEL staining and fluorescence-activated cell sorting (FACS) analysis. SOM230 appears very efficacious in its neuroprotective properties in both models of retinal ischemia affording neuroprotection at the concentration or dose of 100 nM. These data suggest that SOM230 might represent a useful pharmacological compound for the treatment of retinal disease.

Stimulatory effect of SOM230 on human and rat adrenal corticosteroid secretion in vitro.

SOM230 (pasireotide, Signifor), a recently developed somatostatin analog, has been tested in ACTH-secreting pituitary tumors with promising results. No study has yet evaluated whether this analog also directly affects adrenal steroid production. The aim of the current study was to evaluate whether SOM230 modulates corticosteroid secretion by normal adrenals in vitro. Primary cultures from normal human and rat adrenals were incubated with 10-100 nM SOM230 with and without 10nM ACTH. Dose-response studies with 1 nM-1 µM SOM230 were performed on rat adrenals. Cortisol/corticosterone levels in medium were measured after 4 and 24h. SOM230 (10nM) significantly increased corticosteroid levels after 24h incubation in both human (36.4 ± 0.43 ng/well vs 27.7 ± 3.17 ng/well, p<0.05) and rat (16.2 ± 1.16 ng/well vs 11.6 ± 0.92 ng/well p<0.05) adrenals; lesser effects were observed with 100 nM SOM (33.4 ± 2.59 ng/well vs 27.7 ± 3.17 ng/well p<0.05; 13.4 ± 0.82 ng/well vs 11.6 ± 0.92 ng/well, N.S. vs baseline secretion for human and rat adrenals, respectively). Dose-response curves confirmed maximal effect at 10nM SOM230. The corticosteroid secretory response to ACTH was unaffected by SOM230 co-incubation. In conclusion, SOM230 exerts a moderate stimulatory effect on adrenal corticosteroid secretion in vitro. This argues against a direct adrenal involvement in the clinical efficacy of SOM230 in patients with ACTH-secreting pituitary tumors and widens the known range of action of SOM230.

Octreotide and the novel multireceptor ligand somatostatin receptor agonist pasireotide (SOM230) block the adrenalectomy-induced increase in mitotic activity in male rat anterior pituitary.

The novel somatostatin receptor agonist pasireotide binds with high affinity to somatostatin receptors SSTR1, 2, 3, and 5. Acting principally through the latter, it inhibits basal and CRH-stimulated ACTH secretion from the AtT20 corticotroph cell line and ACTH release from a proportion of human corticotroph adenomas both in vitro and in vivo. Data supporting an additional antiproliferative effect has led to pasireotide being explored as a potential therapy for patients with Cushing's disease. We have compared the effects of pasireotide and octreotide on adrenalectomy-induced mitotic and apoptotic activity in the male rat anterior pituitary. Adrenalectomized rats were treated with daily sc injections of vehicle, pasireotide, or octreotide. Changes in proliferation and apoptosis were determined 2-6 d postoperatively. Pasireotide and octreotide had no effect on baseline pituitary cell turnover and no measurable effects on apoptosis. However, the wave of increased mitotic activity normally seen in the pituitary after adrenalectomy was completely abolished. Nevertheless, pasireotide and octreotide did not diminish the increase in ACTH-immunopositive cell index after adrenalectomy, indicating that cell division and differentiation of hormonally null cells in the pituitary are under independent control. In conclusion, basal cell turnover in the pituitary is not inhibited by pasireotide or octreotide. Bilateral adrenalectomy stimulates differentiation of preexisting null cells into ACTH-positive cells. Cell division after bilateral adrenalectomy occurs in a specific subpopulation of hormonally null cells that are equally sensitive to the antiproliferative effects of pasireotide and octreotide, implicating SSTR2 receptors in this antimitotic response.

Inhibitory effect of pasireotide and octreotide on lymphocyte activation.

Somatostatin (SST) regulates the function of the central and peripheral nervous system, the endocrine and exocrine organs, as well as the vascular and immune system. These actions are mediated by five specific membrane somatostatin receptors. This study compares the effects on human lymphocytes of two long-acting somatostatin analogues that have different receptor affinity: octreotide and pasireotide. Both analogues have an antiproliferative effect on human lymphocyte proliferation, but they act at different concentration and, while octreotide enhances IL10 and inhibits gamma IFN pasireotide inhibits IL2 and gamma IFN. In both sets of experiment the different behaviour of the two analogues could be due to their different affinity to the SSTR subtypes. Finally this study suggest that the growth inhibitory action of somatostatin analogues is an apoptotic phenomenon and it can be mediated by SSTR2a, in the case of octreotide, and by SSTR3 when pasireotide is used or it can be mediated by the heterodimerization of the two receptor.

Somatostatin Agonist Pasireotide Inhibits Exercise-Stimulated Growth in the Male Siberian Hamster (Phodopus sungorus).

The Siberian hamster (Phodopus sungorus) is a seasonal mammal, exhibiting a suite of physiologically and behaviourally distinct traits dependent on the time of year and governed by changes in perceived day length (photoperiod). These attributes include significant weight loss, reduced food intake, gonadal atrophy and pelage change with short-day photoperiod as in winter. The central mechanisms driving seasonal phenotype change during winter are mediated by a reduced availability of hypothalamic triiodothyronine (T3), although the downstream mechanisms responsible for physiological and behavioural changes are yet to be fully clarified. With access to a running wheel (RW) in short photoperiod, Siberian hamsters that have undergone photoperiod-mediated weight loss over-ride photoperiod-drive for reduced body weight and regain weight similar to a hamster held in long days. These changes occur despite retaining the majority of hypothalamic gene expression profiles appropriate for short-day hamsters. Utilising the somatostatin agonist pasireotide, we recently provided evidence for an involvement of the growth hormone (GH) axis in the seasonal regulation of bodyweight. In the present study, we employed pasireotide to test for the possible involvement of the GH axis in RW-induced body weight regulation. Pasireotide successfully inhibited exercise-stimulated growth in short-day hamsters and this was accompanied by altered hypothalamic gene expression of key GH axis components. Our data provide support for an involvement of the GH axis in the RW response in Siberian hamsters.

Pasireotide Long-Acting Release Treatment for Diabetic Cats with Underlying Hypersomatotropism.

BACKGROUND: Long-term medical management of hypersomatotropism (HS) in cats has proved unrewarding. Pasireotide, a novel somatostatin analogue, decreases serum insulin-like growth factor 1 (IGF-1) and improves insulin sensitivity in cats with HS when administered as a short-acting preparation. OBJECTIVES: Assess once-monthly administration of long-acting pasireotide (pasireotide LAR) for treatment of cats with HS. ANIMALS: Fourteen cats with HS, diagnosed based on diabetes mellitus, pituitary enlargement, and serum IGF-1 > 1000 ng/mL. METHODS: Uncontrolled, prospective cohort study. Cats received pasireotide LAR (6-8 mg/kg SC) once monthly for 6 months. Fructosamine and IGF-1 concentrations, and 12-hour blood glucose curves (BGCs) were assessed at baseline and then monthly. Product of fructosamine concentration and insulin dose was calculated as an indicator of insulin resistance (Insulin Resistance Index). Linear mixed-effects modeling assessed for significant change in fructosamine, IGF-1, mean blood glucose (MBG) of BGCs, insulin dose (U/kg) and Insulin Resistance Index. RESULTS: Eight cats completed the trial. Three cats entered diabetic remission. Median IGF-1 (baseline: 1962 ng/mL [range 1051-2000 ng/mL]; month 6: 1253 ng/mL [524-1987 ng/mL]; P < .001) and median Insulin Resistance Index (baseline: 812 µmolU/L kg [173-3565 µmolU/L kg]; month 6: 135 µmolU/L kg [0-443 µmolU/L kg]; P = .001) decreased significantly. No significant change was found in mean fructosamine (baseline: 494 ± 127 µmol/L; month 6: 319 ± 113.3 µmol/L; P = .07) or MBG (baseline: 347.7 ± 111.0 mg/dL; month 6: 319.5 ± 113.3 mg/dL; P = .11), despite a significant decrease in median insulin dose (baseline: 1.5 [0.4-5.2] U/kg; 6 months: 0.3 [0.0-1.4] U/kg; P < .001). Adverse events included diarrhea (n = 11), hypoglycemia (n = 5), and worsening polyphagia (n = 2). CONCLUSIONS AND CLINICAL IMPORTANCE: Pasireotide LAR is the first drug to show potential as a long-term management option for cats with HS.

Regulation of CRH-induced secretion of ACTH and corticosterone by SOM230 in rats.

OBJECTIVE: Adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome is biochemically characterized by increased plasma concentrations of ACTH inducing hypersecretion of cortisol. Somatostatin is known to inhibit ACTH secretion, and in vitro data have shown the inhibition of ACTH secretion by agonists activating sst2 and sst5 receptors. The present study aimed to determine the inhibitory effect of the multireceptor ligand SOM230, compared with the sst2-preferring agonist octreotide, on corticotropin-releasing hormone (CRH)-stimulated secretion of ACTH and corticosterone in rats. METHODS: Secretion of ACTH and corticosterone was induced by i.v. application of CRH (0.5 microg/kg) in rats pretreated 1 h before by i.v. application of SOM230 (1, 3, or 10 microg/kg), octreotide (10 microg/kg) or NaCl 0.9%. RESULTS: SOM230 (3 and 10 microg/kg) inhibited CRH-induced ACTH release by 45+/-3% and 51+/-2%, respectively, and corticosterone release by 43+/-5% and 27+/-16%, respectively. 10 microg/kg of octreotide tended to be less potent at inhibiting ACTH release (34+/-6% inhibition) and did not alter the secretion of corticosterone. CONCLUSION: SOM230 has a stronger inhibitory effect on ACTH and corticosterone secretion than octreotide in rats. This difference can be explained by its higher affinity to sst1, sst3 and especially sst5 receptors compared with octreotide.

Pasireotide (SOM230) prevents sulfonylurea-induced hypoglycemia in rats.

Persistent hypoglycemia is a serious condition that is frequently reported in patients undergoing sulfonylurea treatment, often necessitating hospitalization in the event of overdose. Somatostatin is a regulatory hormone with a broad range of physiological actions that include the inhibition of insulin and glucagon secretion, predominantly via activation of the somatostatin receptor subtypes sstr5 and sstr2, respectively. Previous studies have demonstrated that octreotide, a potent somatostatin analogue with high affinity for sstr2 and moderate affinity for sstr5, significantly increases serum glucose levels and prevents recurrence of hypoglycemic episodes in patients with sulfonylurea-induced hypoglycemia. Pasireotide (SOM230) is a multireceptor-targeted somatostatin analogue with a 39-, 30- and 5-fold higher binding affinity for sstr5, sstr1 and sstr3, respectively, and a slightly lower (0.4-fold) affinity for sstr2 compared with octreotide. This study evaluated the effects of pasireotide and octreotide in rats with glyburide-induced hypoglycemia. In fasted rats, pasireotide (10 and 30 µg/kg) prevented glyburide-induced hypoglycemia in a dose-dependent manner for up to 6 h. Qualitatively similar results were observed in non-fasted rats. However, the antihypoglycemic effect of pasireotide was stronger in non-fasted rats, resulting in transient hyperglycemia. In contrast to pasireotide, octreotide 10 µg/kg did not prevent glyburide-induced hypoglycemia in fasted and non-fasted rats, while octreotide 30 µg/kg resulted in small but significant increases in blood glucose at 3 h post-dose only. These findings suggest that pasireotide could have a more potent effect than octreotide in the management of patients with severe hypoglycemia caused by hyperinsulinemia.

Somatostatin Agonist Pasireotide Promotes a Physiological State Resembling Short-Day Acclimation in the Photoperiodic Male Siberian Hamster (Phodopus sungorus).

The timing of growth in seasonal mammals is inextricably linked to food availability. This is exemplified in the Siberian hamster (Phodopus sungorus), which uses the annual cycle of photoperiod to optimally programme energy expenditure in anticipation of seasonal fluctuations in food resources. During the autumn, energy expenditure is progressively minimised by physiological adaptations, including a 30% reduction in body mass, comprising a reduction in both fat and lean tissues. However, the mechanistic basis of this adaptation is still unexplained. We hypothesised that growth hormone (GH) was a likely candidate to underpin these reversible changes in body mass. Administration of pasireotide, a long-acting somatostatin receptor agonist developed for the treatment of acromegaly, to male hamsters under a long-day (LD) photoperiod produced a body weight loss. This comprised a reduction in lean and fat mass, including kidneys, testes and brown adipose tissue, typically found in short-day (SD) housed hamsters. Furthermore, when administered to hamsters switched from SD to LD, pasireotide retarded the body weight increase compared to vehicle-treated hamsters. Pasireotide did not alter photoperiod-mediated changes in hypothalamic energy balance gene expression but altered the expression of Srif mRNA expression in the periventricular nucleus and Ghrh mRNA expression in the arcuate nucleus consistent with a reduction in GH feedback and concurrent with reduced serum insulin-like growth factor-1. Conversely, GH treatment of SD hamsters increased body mass, which included increased mass of liver and kidneys. Together, these data indicate a role for the GH axis in the determination of seasonal body mass of the Siberian hamster.

Pasireotide for the Medical Management of Feline Hypersomatotropism.

BACKGROUND: Feline hypersomatotropism (HST) is a cause of diabetes mellitus in cats. Pasireotide is a novel multireceptor ligand somatostatin analog that improves biochemical control of humans with HST. HYPOTHESIS/OBJECTIVES: Pasireotide improves biochemical control of HST and diabetes mellitus in cats. ANIMALS: Hypersomatotropism was diagnosed in diabetic cats with serum insulin-like growth factor-1 (IGF-1) concentration >1,000 ng/mL by radioimmunoassay and pituitary enlargement. METHODS: Insulin-like growth factor 1 was measured and glycemic control assessed using a 12-hour blood glucose curve on days 1 and 5. On days 2, 3, and 4, cats received 0.03 mg/kg pasireotide SC q12h. IGF-1, insulin dose, and estimated insulin sensitivity (product of the area under the blood glucose curve [BGC] and insulin dose) were compared pre- and post treatment. Paired t-tests or Wilcoxon signed rank tests were employed for comparison where appropriate; a linear mixed model was created to compare BGC results. RESULTS: Insulin-like growth factor 1 decreased in all 12 cats that completed the study (median [range] day 1: 2,000 ng/mL [1,051-2,000] and day 5: 1,105 ng/mL [380-1,727], P = .002, Wilcoxon signed rank test). Insulin dose was lower on day 5 than on day 1 (mean reduction 1.3 [0-2.7] units/kg/injection, P = .003, paired t-test). The product of insulin dose and area under the BGC was lower on day 5 than day 1 (difference of means: 1,912; SD, 1523; u × mg/dL × hours, P = .001; paired t-test). No clinically relevant adverse effects were encountered. CONCLUSIONS: Short-acting pasireotide rapidly decreased IGF-1 in cats with HST and insulin-dependent diabetes. The decrease in IGF-1 was associated with increased insulin sensitivity.

Inhibition of the nicotinic acetylcholine response by serotonergic and muscarinic agents in chick ciliary ganglion neurones.

Chick ciliary ganglion neurones were investigated by whole cell voltage clamp recordings. The ACh- or nicotine-induced inward current was partially inhibited by perfusing the neurones with 5-HT. This effect was rapid (< or = 1 min), dose-dependent (50-1000 microM) and quickly reversible. The selective 5-HT1A agonist 8-OH-DPAT (10 microM) reduced the nicotinic ACh response more potently, irrespective of the absence or presence of propranolol (1 microM), a known 5-HT1A antagonist. Other serotonergic antagonists, like ICS 205-930 (1 microM), mianserin (10 microM) and methysergide (10 microM), also failed to antagonize the 5-HT-mediated decrease in the nicotinic response. Muscarine (50 microM) did not affect the nicotine-induced inward current but the muscarinic agonist oxotremorine (10 microM) also decreased the nicotine-induced inward current. Atropine, at small concentrations failed to block this effect but caused some reduction of the ACh response itself at larger (1-10 microM) concentrations. It is suggested that 5-HT may modulate synaptic transmission in ciliary ganglion neurones in vivo. The site of action of 5-HT, oxotremorine and atropine might be at or close to the ACh receptor complex, because of the fast onset and reversibility of the effects and lack of specificity for structurally different drugs.

Electrophysiological responses of neurons in the rat spinal cord to nitric oxide.

The effects of nitric oxide-containing solution and different nitric oxide donors were investigated on spontaneously active neurons using extracellular recording technique in areas of rat spinal cord slices where high levels of nitric oxide synthase are present. In lamina X, 93% of all neurons investigated (n = 84) increased their firing rate and 2% decreased it by superfusion with the nitric oxide donor sodium nitroprusside. In contrast, 49% of all neurons in laminae I and II (n = 90) were inhibited and only 28% were activated. Both effects were due to the postsynaptic action of sodium nitroprusside, because they could still be observed in medium containing 0.3 mM Ca2+ and 9 mM Mg2+, known to block synaptic transmission. Application of 8-bromo-cyclic-GMP caused an excitation of every neuron which was excited by sodium nitroprusside and an inhibition of every cell which was inhibited by sodium nitroprusside (n = 25). This effect was different from the effect of 8-bromo-cyclic-AMP, which mimicked only the excitatory, but not the inhibitory response of sodium nitroprusside. These results provide evidence that nitric oxide in the spinal cord can directly cause an excitation or an inhibition of the electrical activity of spinal neurons. Another, more general conclusion from our results is that the nitric oxide-induced production of cyclic-GMP alone does not allow any prediction about an excitatory or inhibitory effect on the neuronal activity, which has to be determined separately.

Vasopressin and sensory circumventricular organs.

The subfornical organ, the area postrema and the organum vasculosum of the lamina terminalis are considered to be sensory circumventricular organs as they contain neuronal somata which are located outside the blood-brain barrier and are thus capable of serving as 'sensors' for blood-borne humoral messengers. The endocrine hormone, vasopressin (VP), not only causes strong antidiuresis by acting on the kidney, but also exerts centrally mediated effects as a neuromodulator. Several lines of evidence suggest that VP can influence regulatory functions mediated by the sensory circumventricular organs, since vasopressinergic somata and terminals as well as VP receptors have been reposted to be present in these structures. These biochemical prerequisites offer the possibility that blood-borne VP might on the one hand act as a feedback signal from the periphery and, on the other hand, synaptically released or locally produced VP could modulate the known functions of sensory circumventricular organs, such as thirst, fever or cardiovascular regulation. This review focuses on the possible physiological relevance of VP acting on sensory circumventricular organs in view of recent evidence obtained from biochemical and electrophysiological studies at the cellular level.

Spinal neuronal thermosensitivity in vivo and in vitro in relation to hypothalamic neuronal thermosensitivity.

In the spinal cord, temperature signals are generated which serve as specific inputs in the central nervous control of body temperature. Because of the spatially distinct organization of afferent and efferent neuronal systems at the spinal level, the afferent pathway for temperature signal transmission could be identified in vivo in the ascending, anterior and lateral tracts with a relationship of about 75:25% between warm and cold sensitive neuraxons. Analysis of spinal neuronal thermosensitivity in vitro on spinal cord tissue slices has been concerned, so far, with the superficial laminae of the dorsal horn as the site of origin of ascending nerve fibers conveying mostly temperature and pain signals, and with lamina X as a site of origin of afferent as well as efferent neurons. A relationship of about 95:5% between warm and cold sensitive neurons was found at the segmental level, indicating that warm sensitivity is the prevailing, primary property of spinal neurons, whereas cold sensitivity seems to be mainly generated by synaptic interaction as a secondary modality. Dynamic responses to temperature changes were frequently displayed in vitro at the spinal segmental level in lamina I + II but not in lamina X, even by neurons whose static activity was little influenced by local temperature. Dynamic thermosensitivity was found less frequently in ascending tract neuraxons and was not observed in hypothalamic neurons receiving temperature signal inputs from the spinal cord, and thus, does not seem to be relevant for the thermosensory function of spinal cord neurons, unlike peripheral warm and cold receptors. A majority of spinal warm sensitive neurons displayed both static and dynamic warm sensitivity as an inherent property after synaptic blockade. In the further analysis of spinal cord thermosensitivity, the in vitro approach permits application of the same electrophysiological and neuropharmacological methods as were established for the analysis of hypothalamic thermosensitivity. In addition, the topography of the spinal cord will provide additional structural and possibly histochemical information to characterize the functions of neurons independently of their thermal properties.

Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus.

Leptin decreases food intake and increases energy expenditure in rodents by inhibiting neurones in the hypothalamic arcuate nucleus. The growth hormone secretagogue (GHS) ghrelin is known to stimulate food intake and to be the endogenous ligand for the GHS-receptor, which is strongly expressed in the arcuate nucleus, like the leptin receptor (Ob-R). In this study, we analysed the effect of systemic ghrelin administration on Fos expression in the arcuate nucleus on neurones expressing Ob-R. Injection of ghrelin (0.2 mg/kg, i.p) significantly increased the number of neurones expressing Fos protein in the ventromedial arcuate nucleus. Fifty-seven percent of all Fos-positive cells in the ventromedial arcuate nucleus were also positive for Ob-R staining. Furthermore, we investigated electrophysiologically the effect of ghrelin and leptin on the activity of arcuate neurones in an in-vitro slice preparation. Ghrelin stimulated the electrical activity dose-dependently in 80% of all cells tested (n=49) with a threshold concentration of 10(-11) M; only 8% were inhibited and 12% did not respond. The effect of ghrelin (10(-7) M) was weakly antagonized by the peptidic GHS-receptor antagonist (D-Lys3)-GHRP-6 (10(-4) M), which also showed a much weaker affinity (IC(50), 0.9 x 10(-6) M) to the GHS-receptor than ghrelin (IC(50), 0.3 x 10(-9) M). Ghrelin increased the electrical activity in 76% of all cells which were inhibited by leptin (n=17). These data show that ghrelin interacts with the leptin hypothalamic network in the arcuate nucleus. The opposite effect of leptin and ghrelin on neurones in the arcuate nucleus may serve as a neurophysiological correlate of the orexigenic and anorectic effects of ghrelin and leptin.

Regional specific effects of nitric oxide donors and cGMP on the electrical activity of neurons in the rat spinal cord.

Numerous functional studies establish the role of nitric oxide (NO) as a neuromodulator in the central nervous system which affects synaptic transmission. However, there are only a few reports indicating a direct and postsynaptic effect of nitric oxide on the electrical activity of neurons in the central nervous system. The aim of this study was to characterize the effect of nitric oxide on spontaneously active neurons in spinal cord slices using an extracellular recording technique. Because in the lumbar rat spinal cord the NO producing enzyme NO-synthase is primarily located in the superficial dorsal horn (laminae I+II) and around the central canal (lamina X), we restricted our recordings to these areas. While the majority of neurons increased their electrical activity during superfusion with the NO-donor sodium nitroprusside (SNP) in lamina X, neurons in laminae I+II were mainly inhibited by SNP. The excitatory and the inhibitory effects were dose-dependent and reversible and were mimicked by other NO-donors and membrane permeable cyclic guanosine monophosphate (8Br-cGMP) on the same neurons. The spinal cord slice preparation contains functional NO-synthase (NOS), because selective blockade of NOS increased the spontaneous activity of those neurons from laminae I+II which were inhibited by SNP and this effect could be reversed by superfusion with the natural substrate for NOS, L-arginine. It is concluded that NO can activate and inhibit the activity of spinal cord neurons by raising cGMP levels and that these effects are lamina specific. A general consequence of our results is that the NO-induced production of cGMP alone does not allow any prediction about an excitatory or inhibitory effect of NO on the discharge rate of neurons. Thus the NO mediated increase and decrease in neuronal activity is probably the result of intracellular mechanisms downstream from the production of cGMP which results in the activation or inhibition of different ion channels on neurons in laminae I+II and X.

Effect of angiotensin II and atrial natriuretic factor on neurons in the subfornical organ of ducks and rats in vitro.

Atrial natriuretic factor (ANF) antagonizes many angiotensin II (ANGII)-induced effects on osmoregulatory relevant parameters in vivo. In this study ANF analogues decreased the spontaneous and the ANGII-induced electrical activity of subfornical organ (SFO) neurons in rats, but had no effect on ANGII sensitive or insensitive SFO neurons in ducks. These results suggest a more distinct functional separation for the responsiveness to ANGII and ANF in birds compared to mammals.

Primary culture of circumventricular organs from the rat brain lamina terminalis.

A primary culture system of cells derived from two circumventricular organs (CVO) of the rat brain was established. The subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) were dissected from the rostral wall of the third ventricle and its cells taken into culture after mechanical dissociation. The cells were cultured in a modified microculture chamber system ensuring relatively high cell density despite their low absolute number. When animals were injected with Evans blue prior to cell preparation, the macroscopically visible penetration of the dye into the parenchyma of the CVOs could be used as guidance during tissue isolation and labelled cells could be identified in culture. Cultured CVO neurones and astrocytes were identified using antibodies against cell type specific marker proteins. The histochemical NADPH-diaphorase staining was used for the detection of nitric oxide synthase in tissue sections of both CVOs and in their cultured neurones. In addition, angiotensin II (ANG II)-evoked elevations of the intracellular Ca2+ concentration ([Ca2+]i) in single cultured OVLT neurones were measured. The described methods will be useful for further characterization of CVO neurones and astrocytes.

Pharmacological characterisation of amylin-related peptides activating subfornical organ neurones.

Amylin, calcitonin gene-related peptide (CGRP) and calcitonin are structurally related peptides with overlapping peripheral and central actions. Amylin and calcitonin excite the majority of neurones in the subfornical organ (SFO), where high densities of so-called C-type G-protein-coupled receptors have been detected. Subcutaneous injection of these hormones stimulates drinking similar to angiotensin II (ANGII), a dipsogen acting via the SFO. We now show that in addition to amylin and rat calcitonin (rCT), CGRP and salmon calcitonin (sCT) also excite SFO neurones. In extracellular recordings of an in vitro slice preparation of the SFO, 78% of all neurones (n=31) superfused with CGRP (10(-6) M) were excited. The excitatory effect was dose-dependent and reversible with an average threshold concentration of 5x10(-7) M, which is approximately 15-fold higher than reported for amylin-induced excitations. sCT (10(-7) M), which behaves as a non-competitive agonist at amylin as well as calcitonin receptors, caused irreversible excitatory responses in 96% of all recordings (n=26). Amylin-, CRGP- and rCT-induced excitations could be blocked by the selective amylin receptor antagonist AC187 (10(-5) to 10(-6) M), whereas sCT-induced excitations were not inhibited. The receptor antagonist human CGRP(8-37) (10(-6) M) partly caused agonistic responses, but did not block CGRP-induced excitations. The pharmacological profile observed in the present work, and in a recent publication using the same preparation, indicating (1) that CGRP is a weaker agonist in the SFO than amylin, (2) that sCT excites SFO neurones, and (3) that responses are blocked by AC187 but not by CGRP(8-37), is inconsistent with activation via CGRP receptors, but is instead consistent with involvement of amylin (C3) and calcitonin (C1) receptors, which are co-localized to a high degree on the same subset of SFO-neurones. We propose that it is unlikely that blood-borne CGRP has a significant effect on neurones in the SFO.

Angiotensin II receptor subtypes in the duck subfornical organ: an electrophysiological and receptor autoradiographic investigation.

The pharmacology of angiotensin II (AngII) receptors was investigated in the brain of ducks using receptor autoradiographic and electrophysiological methods. Using 125I[Val5]AngII as a ligand, specific binding was observed in sections of the duck adrenal gland and in several brain areas involved in body fluid homeostasis. Displacement studies using the same antagonists as used for classifying mammalian AngII receptor subtypes revealed that the rank order of potencies in competition with AngII receptors in the adrenal gland and in the subfornical organ was: AngII > CGP-42112A > losartan > PD-123319. Electrophysiological recordings from spontaneously active neurons of duck SFO slices revealed that the majority of neurons could be excited by AngII (10(-7) M). The excitatory effect of AngII could be partially inhibited by CGP-42112A (10(-5) M), which proved to be more effective than equimolar losartan and far more effective than PD-123319. These data suggest that the neuronal AngII receptors in the SFO are pharmacologically distinct from the mammalian AT1- and AT2-receptors. Further, central AngII receptors of ducks share common pharmacological characteristics with AngII receptors in the duck adrenal gland and peripheral organs of other bird species.