Neuronostatin is co-expressed with somatostatin and mobilizes calcium in cultured rat hypothalamic neurons.

Neuronostatin (NST) is a newly identified peptide of 13-amino acids encoded by the somatostatin (SST) gene. Using a rabbit polyclonal antiserum against the human NST, neuronostatin-immunoreactive (irNST) cells comparable in number and intensity to somatostatin immunoreactive (irSST) cells were detected in the hypothalamic periventricular nucleus. Fewer and/or less intensely labeled irNST cells were noted in other regions such as the hippocampus, cortex, amygdala, and cerebellum. Double-labeling hypothalamic sections with NST- and SST-antiserum revealed an extensive overlapping of irNST and irSST cells in the periventricular nucleus. Pre-absorption of the NST-antiserum with NST (1 microg/ml) but not with SST (1 microg/ml) abrogated irNST and vice versa. The activity of NST on dissociated and cultured hypothalamic neurons was assessed by the Ca(2+) imaging method. NST (10, 100, 1000 nM) concentration-dependently elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in a population of hypothalamic neurons with two distinct profiles: (1) a fast and transitory increase in [Ca(2+)](i), and (2) an oscillatory response. Whereas, SST (100 nM) reduced the basal [Ca(2+)](i) in 21 of 61 hypothalamic neurons examined; an increase was not observed in any of the cells. Optical imaging with a slow-responding voltage sensitive dye DiBAC(4)(3) showed that NST (100 nM) depolarized or hyperpolarized; whereas, SST (100 nM) hyperpolarized a population of hypothalamic neurons. The result shows that NST and SST, though derived from the same precursor protein, exert different calcium mobilizing effects on cultured rat hypothalamic neurons, resulting in diverse cellular activities.

C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.

With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.

Copeptin immunoreactivity and calcium mobilisation in hypothalamic neurones of the rat.

Copeptin is cleaved from the C-terminus of vasopressin (VP) prohormone. Immunohistochemical studies have revealed intense copeptin-immunoreactivity (irCOPT) in neurones of the rat hypothalamic nuclei, including paraventricular, supraoptic, suprachiasmatic, periventricular, and accessory secretory. Varicose cell processes emanated from irCOPT neurones, some of which projected caudally and traversed the internal layer of the median eminence, and terminated in the posterior pituitary. Double-labelling hypothalamic sections with copeptin antiserum and VP or oxytocin antiserum revealed an extensive overlapping of irCOPT and irVP neurones. The biological activity of human synthetic nonglycosylated copeptin or VP was evaluated in vivo and in vitro. Copeptin (1, 10, and 20 nmol/kg) injected i.v. caused no significant changes in the mean arterial pressure (MAP) and heart rate of urethane-anaesthetised rats. VP (0.1 nmol/kg) increased MAP, which was accompanied by a small decrease of the heart rate. The ratiometric fluorescence method was employed to assess changes in intracellular Ca2+ concentrations [Ca2+](i) which served as an index of the biological activity of peptides. VP (1 microM) markedly increased [Ca2+](i) of rat hypothalamic neurones or vascular smooth muscle cells, whereas copeptin (100 nm to 1 microM) caused a low amplitude, sustained increase of [Ca2+](i) in a population of hypothalamic neurones, but not in any of the vascular smooth muscle cells tested. The results obtained demonstrate that copeptin is expressed in VP neurones and that the peptide in the concentrations tested, although causing little or no detectable changes of blood pressure and heart rate in anaesthetised rats nor changes in [Ca2+](i) of cultured aortic smooth muscle cells, increases [Ca2+](i) in a small population (< 2%) of hypothalamic neurones tested, indicating that copeptin is biologically active in mammalian neurones.

Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons.

Human immunodeficiency virus 1 (HIV-1) Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current- or voltage-clamp mode. Tat30-86 (50-1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 microM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) and d-2-amino-5-phosphonovaleric acid (AP-5) (50 microM), and were markedly reduced in a Ca(2+)-free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between -30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca(2+) imaging studies showed that Tat30-86 produced three types of Ca(2+) responses: 1) a fast and transitory increase, 2) Ca(2+) oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca(2+) response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons.

Beacon immunoreactivity in the rat hypothalamus.

Beacon (BC) is a peptide of 73 amino acids, whose gene expression was first reported in the hypothalamus of Psammomys obesus (or Israeli sand rat). To appreciate better the functional role of BC in normal rats and sand rats, the distribution of BC immunoreactivity (irBC) and its subcellular localization were studied in the brain of Sprague-Dawley rats. In the hypothalamus, intense staining was present in neurons of the supraoptic (SO), paraventricular (PVH), and accessory neurosecretory nuclei and in cell processes of median eminence. Double labeling of the hypothalamic sections with mouse monoclonal oxytocin (OT) antibody and rabbit polyclonal BC antiserum revealed that nearly all OT-immunoreactive cells from SO, PVH, and accessory neurosecretory nuclei were irBC. Double labeling of the sections with guinea pig vasopressin (VP) antiserum and BC antiserum showed that a population of VP-immunoreactive neurons was irBC. By immunoelectron microscopy, immunoreactive product was associated with mitochondrial membranes or appeared as electron-dense bodies in many PVH and SO neurons. Most of the neurosecretory granules were unstained for BC. Taken together, our results indicate the presence of beacon in the OT-containing neurons and a population of VP-containing neurons, mostly associated with mitochondrial membrane. Insofar as the amino acids sequence of beacon is identical to that of ubiquitin-like 5, it is possible that the distribution of BC immunoreactivity noted in our study is that of ubiquitin-like 5 peptide in the rat hypothalamus.

The vasoactive peptide urotensin II stimulates spontaneous release from frog motor nerve terminals.

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca(2+) three-fold had no effect on the MEPP frequency increase to 25 microM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca(2+) caused a 61% reduction in the MEPP frequency increase to 25 microM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 microM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP(3)) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 microM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP(3) and cyclic AMP/protein kinase A.

Sympathoinhibitory action of nociceptin in the rat spinal cord.

1. Whole-cell patch recordings were made from antidromically identified sympathetic preganglionic neurons (SPN) of immature rat spinal cord slices. Bath application of nociceptin (0.1-1 micromol/L) suppressed excitatory postsynaptic potentials (EPSP) and hyperpolarized a population of SPN; these effects were naloxone (1 micromol/L) insensitive. 2. Nociceptin suppressed the amplitude of EPSP without causing a concomitant change in glutamate-induced depolarizations, suggesting a presynaptic inhibitory action. 3. Analysis of current-voltage relationships showed that nociceptin hyperpolarized SPN by increasing an inwardly rectifying K+ current. 4. Intrathecal injection of nociceptin (3, 10 and 30 nmol) to urethane-anaesthetized rats dose-dependently reduced the mean arterial pressure and heart rate; these effects were not prevented by prior intravenous injection of naloxone (1 mg/kg). 5. Results from our in vitro and in vivo experiments suggest that nociceptin suppresses spinal sympathetic outflow either by attenuating excitatory synaptic responses or hyperpolarizing SPN.

Orexins/hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro.

The two recently isolated hypothalamic peptides orexin A and orexin B, also known as hypocretin 1 and 2, are reported to be important signaling molecules in feeding and sleep/wakefulness. Orexin-containing neurons in the lateral hypothalamus project to numerous areas of the rat brain and spinal cord including the intermediolateral cell column (IML) of the thoracolumbar spinal cord. An in vivo and in vitro study was undertaken to evaluate the hypothesis that orexins, acting on sympathetic preganglionic neurons (SPNs) in the rat spinal cord, increase sympathetic outflow. First, orexin A (0.3, 1, and 10 nmol) by intrathecal injection increased mean arterial pressure (MAP) and heart rate (HR) by an average of 5, 18, and 30 mmHg and 10, 42, and 85 beats/min in urethane-anesthetized rats. Intrathecal injection of saline had no significant effects. Orexin B (3 nmol) by intrathecal administration increased MAP and HR by an average of 11 mmHg and 40 beats/min. The pressor effects of orexin A were attenuated by prior intrathecal injection of orexin A antibodies (1:500 dilution) but not by normal serum albumin. Intravenous administration of the alpha(1)-adrenergic receptor antagonist prazosin (0.5 mg/kg) or the beta-adrenergic receptor antagonist propranolol (0.5 mg/kg) markedly diminished, respectively, the orexin A-induced increase of MAP and HR. Second, whole cell patch recordings were made from antidromically identified SPNs of spinal cord slices from 12- to 16-day-old rats. Superfusion of orexin A or orexin B (100 or 300 nM) excited 12 of 17 SPNs, as evidenced by a membrane depolarization and/or increase of neuronal discharges. Orexin A- or B-induced depolarizations persisted in TTX (0.5 microM)-containing Krebs solution, indicating that the peptide acted directly on SPNs. Results from our in vivo and in vitro studies together with the previous observation of the presence of orexin A-immunoreactive fibers in the IML suggest that orexins, when released within the IML, augment sympathetic outflow by acting directly on SPNs.

Urotensin II-immunoreactivity in the brainstem and spinal cord of the rat.

The distribution of urotensin-II-immunoreactivity (irU-II) was studied in the rat brainstem and spinal cord with the use of an antiserum against the human urotensin II (U-II) peptide. A population of ventral horn neurons in the spinal cord, hypoglossal nucleus, dorsal motor nucleus of the vagus, facial motor nucleus, nucleus ambiguus, abducens nucleus and trigeminal motor nucleus exhibited irU-II of varying intensities. The number of irU-II motor neurons was higher in the lumbar segments as compared to that of cervical, thoracic and sacral segments. Double-labeling the sections with U-II- and choline acetyltransferase (ChAT)-antisera revealed that nearly all irU-II ventral horn and brainstem neurons were ChAT-positive. The result provides the first immunohistochemical evidence of the presence of irU-II in cholinergic motoneurons of the rat spinal cord and brainstem.