Eugen Brailoiu

KiSS-1 expression and metastin-like immunoreactivity in the rat brain

Metastin, the gene product of metastasis suppressor gene KiSS‐1, is the endogenous ligand for the G‐protein‐coupled receptor GPR54 (or AXOR12, or OT7T175). The expression of KiSS‐1 gene and peptide and the distribution of metastin were studied in the rat central nervous system by reverse transcriptase‐polymerase chain reaction, Western blotting, and immunohistochemical methods. KiSS‐1 gene and peptide expression was higher in the hypothalamus than in the brainstem and spinal cord. In the brain, metastin‐like immunoreactivity (irMT) was found mainly in three groups of cells: dorsomedial hypothalamic nucleus, nucleus of the solitary tract, and caudal ventrolateral medulla. Immunoreactive fibers of varying density were noted in bed nucleus of stria terminalis, septal nuclei, nucleus accumbens, caudate putamen, diagonal band, amygdala, hypothalamus, zona incerta, thalamus, periaqueductal gray, raphe nuclei, lateral parabrachial nucleus, locus coeruleus, spinal trigeminal tract, rostral ventrolateral medulla, and medullary reticular nucleus. Preabsorption of the antiserum with metastin peptide fragment (45–54)‐NH2 (1 μg/ml) resulted in no staining in any of the sections. The biological activity of metastin was assessed by monitoring intracellular calcium [Ca2+]i in cultured hippocampal neurons, which are known to express GPR54. Metastin increased [Ca2+]i in a population of cultured hippocampal neurons. The results show that metastin is biologically active in rat central neurons, and its anatomical distribution suggests a possible role in nociception and autonomic and neuroendocrine functions. J. Comp. Neurol. 481:314–329, 2005.

Modulation of spontaneous transmitter release from the frog neuromuscular junction by interacting intracellular Ca2+ stores: critical role for nicotinic acid-adenine dinucleotide phosphate (NAADP)

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a recently described potent intracellular Ca(2+)-mobilizing messenger active in a wide range of diverse cell types. In the present study, we have investigated the interaction of NAADP with other Ca(2+)-mobilizing messengers in the release of transmitter at the frog neuromuscular junction. We show, for the first time, that NAADP enhances neurosecretion in response to inositol 1,4,5-trisphosphate (IP(3)), cADP-ribose (cADPR) and sphingosine 1-phosphate (S1P), but not sphingosylphosphorylcholine. Thapsigargin was without effect on transmitter release in response to NAADP, but blocked the responses to subsequent application of IP(3), cADPR and S1P and their potentiation by NAADP. Asynchronous neurotransmitter release may therefore involve functional coupling of endoplasmic reticulum Ca(2+) stores with distinct Ca(2+) stores targeted by NAADP.

Urotensin-II regulates intracellular calcium in dissociated rat spinal cord neurons

Urotensin‐II (U‐II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U‐II on [Ca2+]i was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase‐positive and had morphological features of motoneurons. U‐II induced [Ca2+]i increases in these neurons with a threshold of 10−9u2003m, and a maximal effect at 10−6u2003m with an estimated EC50 of 6.2u2003×u200310−9u2003m. The [Ca2+]i increase induced by U‐II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+‐free medium. Omega‐conotoxin GVIA (10−7u2003m), a N‐type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega‐conotoxin GVIIC (10−7u2003m) and the l‐type Ca2+ channel blocker, verapamil (10−5u2003m) had minimal effects. Down‐regulation of protein kinase C by 4‐α‐phorbol 12‐myristate 13‐acetate (10−6u2003m) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10−6u2003m) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10−6u2003m) or Rp‐8‐pCPT‐cGMPS (3u2003×u200310−5u2003m) did not modify U‐II‐induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10−6u2003m) and Rp‐cAMPS (3u2003×u200310−5u2003m) reduced the response to 25u2003±u20033% and 42u2003±u20038%, respectively. Present results demonstrate that U‐II modulates [Ca2+]i in rat spinal cord neurons via protein kinase A cascade.

Contractile effects by intracellular angiotensin II via receptors with a distinct pharmacological profile in rat aorta.

We studied the effect of intracellular angiotensin II (Ang II) and related peptides on rat aortic contraction, whether this effect is pharmacologically distinguishable from that induced by extracellular stimulation, and determined the Ca2+ source involved. Compounds were delivered into the cytoplasm of de‐endothelized aorta rings using multilamellar liposomes. Contractions were normalized to the maximum obtained with phenylephrine (10−5u2003M). Intracellular administration of Ang II (incorporation range: 0.01–300u2003nmolu2003mg−1) resulted in a dose‐dependent contraction, insensitive to extracellular administration (10−6u2003M) of the AT1 receptor antagonist CV11947, the AT2 receptor antagonist PD 123319, or the non‐selective AT receptor antagonist and partial agonist saralasin ([Sar1,Val5,Ala8]‐Ang II (P<0.05). Intracellular administration of CV11947 or PD 123319 right shifted the dose‐response curve about 1000 fold or 20 fold, respectively. PD 123319 was only effective if less than 30u2003nmolu2003mg−1 Ang II was incorporated. Contraction was partially desensitized to a second intracellular Ang II addition after 45u2003min (P<0.05). Intracellular administration of Ang I and saralasin also induced contraction (P<0.05). Both responses were sensitive to intracellular CV11947 (P<0.05), but insensitive to PD 123319. The response to Ang I was independent of intracellular captopril. Contraction induced by extracellular application of Ang II and of Ang I was abolished by extracellular pre‐treatment with saralasin or CV11947 (P<0.05), but not with PD 123319. Extracellular saralasin induced no contraction. Intracellular Ang II induced contraction was not affected by pre‐treatment with heparin filled liposomes, but completely abolished in Ca2+‐free external medium. These results support the existence of an intracellular binding site for Ang II in rat aorta. Intracellular stimulation induces contraction dependent on Ca2+‐influx but not on Ins(1,4,5)P3 mediated release from intracellular Ca2+‐stores. Intracellular Ang I and saralasin induce contraction, possibly via the same binding site. Pharmacological properties of this putative intracellular receptor are clearly different from extracellular stimulated AT1 receptors or intracellular angiotensin receptors postulated in other tissue.

Sphingosine 1‐phosphate enhances spontaneous transmitter release at the frog neuromuscular junction

Intracellular recordings were made from isolated frog sciatic‐sartorius nerve‐muscle preparations, and the effects of sphingosine 1‐phosphate (S1‐P) on miniature endplate potentials (MEPPs) were studied. Extracellular application of S1‐P (1 and 30 μM) had no significant effects on the frequency and amplitude of MEPPs. Delivery into nerve terminals by liposomes containing 10−5, 10−4 or 10−3 M S1‐P was associated with a concentration‐dependent increase in MEPP frequency of 37, 63 and 86%. The per cent of median MEPP amplitude was not significantly changed, but there was an increase in the number of ‘giant’ MEPPs. Pre‐exposure of the preparations to S1‐P 10−5 but not 10−8 M entrapped in liposomes for 15 min blocked the effects of subsequent superfusion of S1‐P (10−4 M)‐filled liposomes on MEPP frequency. Thus, intracellular S1‐P receptors seem to undergo ‘desensitization’ to higher concentrations of S1‐P. The result provides the first evidence that S1‐P acting intracellularly but not extracellularly enhances spontaneous transmitter release at the frog neuromuscular junction.

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

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. 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. Increasing extracellular Ca2+ three‐fold had no effect on the MEPP frequency increase to 25 μM hU‐II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca2+ caused a 61% reduction in the MEPP frequency increase to 25 μM hU‐II. Pretreatment with the phospholipase C inhibitor U‐73122 caused a 93% reduction in the MEPP frequency increase to 25 μM hU‐II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5‐trisphosphate (IP3) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 μM 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. Our results show that hU‐II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP3 and cyclic AMP/protein kinase A.

Ultra low concentrations of morphine increase neurite outgrowth in cultured rat spinal cord and cerebral cortical neurons.

The present study was undertaken to evaluate the effects of ultra low concentrations (10(-9) or 10(-14)M) of morphine on neurite elongation in cultured neurons dissociated from rat spinal cords and cerebral cortex. In fetal serum (FS) or fetal serum-free supplemented with cAMP media, the length of longest neurite was significantly increased by 10(-9) or 10(-14)M morphine. For example, 10(-14)M morphine increased neurite length by 24 +/- 0.5% and 27 +/- 0.3% in spinal cord neurons, and 18 +/- 0.2% and 17 +/- 0.6% in cortical neurons. Morphine (10(-6)M) had no significant effect on neurite length of spinal and cortical neurons. The relative frequency distribution of neurite length revealed 61 +/- 2.7% of spinal neurons and 48 +/- 2.6% of cortical neurons are responsive to ultra low concentrations of morphine. In the responsive populations, morphine (10(-14)M) enhanced the neurite outgrowth in spinal neurons by 58 +/- 0.9% and 48 +/- 1.2% and in cortical neurons by 31 +/- 0.6% and 28 +/- 0.9% in FS and cAMP-supplemented media, respectively. Pretreatment with naloxone did not prevent the morphine effect. The result shows that morphine at ultra low concentrations enhances neurite outgrowth of spinal and cortical neurons via a naloxone-independent mechanism.

Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth muscle cells.

Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin II is unclear. Therefore, we investigated the effects of intracellular angiotensin II in cells devoid of physiological responses to extracellular angiotensin II (A7r5 vascular smooth muscle cells). Intracellular delivery of angiotensin II was obtained by using liposomes or cell permeabilisation. Intracellular angiotensin II stimulated Ca2+ influx, as measured by 45Ca2+ uptake and single-cell fluorimetry. This effect was insensitive to extracellular or intracellular addition of losartan (angiotensin AT(1) receptor antagonist) or PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) (angiotensin AT2 receptor antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable 45Ca2+ pool in permeabilised cells, independent of losartan and PD123319. Small G-proteins did not participate in this process, as assessed by using GDPbetaS. Intracellular delivery of angiotensin I was unable to elicit any of the effects elicited by intracellular angiotensin II. We conclude from our intracellular angiotensin application experiments that angiotensin II modulates Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological properties suggest the involvement of putative angiotensin non-AT1-/non-AT2 receptors.

Effects of m-CPP in Altering Neuronal Function: Blocking Depolarization in Invertebrate Motor and Sensory Neurons but Exciting Rat Dorsal Horn Neurons

The compound m-chlorophenylpiperazine (m-CPP) is used clinically to manipulate serotonergic function, though its precise mechanisms of actions are not well understood. m-CPP alters synaptic transmission and neuronal function in vertebrates by non-selective agonistic actions on 5-HT(1) and 5-HT(2) receptors. In this study, we demonstrated that m-CPP did not appear to act through a 5-HT receptor in depressing neuronal function in the invertebrates (crayfish and Drosophila). Instead, m-CPP likely decreased sodium influx through voltage-gated sodium channels present in motor and primary sensory neurons. Intracellular axonal recordings showed that m-CPP reduced the amplitude of the action potentials in crayfish motor neurons. Quantal analysis of excitatory postsynaptic currents, recorded at neuromuscular junctions (NMJ) of crayfish and Drosophila, indicated a reduction in the number of presynaptic vesicular events, which produced a decrease in mean quantal content. m-CPP also decreased activity in primary sensory neurons in the crayfish. In contrast, serotonin produces an increase in synaptic strength at the crayfish NMJ and an increase in activity of sensory neurons; it produces no effect at the Drosophila NMJ. In the rat spinal cord, m-CPP enhances the occurrence of spontaneous excitatory postsynaptic potentials with no alteration in evoked currents.

Inositol derivatives modulate spontaneous transmitter release at the frog neuromuscular junction

One of the consequences of G-protein-coupled receptor activation is stimulation of phosphoinositol metabolism, leading to the generation of IP3 and its metabolites 1,3,4,5-tetrakisphosphate (IP4) and inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Previous reports indicate that high inositol polyphosphates (IP4 and IP6) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP4 and IP6 on spontaneous transmitter release in the form of miniature endplate potentials (MEPP) and on enhanced vesicular recycling by high K+ at frog motor nerve endings. In resting conditions, IP4 and IP6 delivered intracellularly via liposomes, caused concentration-dependent increases in MEPP frequency and amplitude. Pretreatment with the protein kinase A (PKA) inhibitor H-89 or KT 5720 reduced the IP4-mediated MEPP frequency increase by 60% and abolished the IP6-mediated MEPP frequency increases as well as the enhancement in MEPP amplitude. Pretreatment with antibodies against phosphatidylinositol 3-kinase (PI 3-K), enzyme also associated with clathrin-coated vesicular recycling, did not alter the IP4 and IP6-mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP3, but not other second messengers releasing Ca2+ from internal Ca2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca2+ release vs. metabolism to IP4 and IP6, we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP3), which is not converted to IP4 or IP6. 3F-IP3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K+-Ringer solution, IP4 and IP6 have similar effects, except decreasing MEPP frequency at a higher concentration (10(-4) M). We conclude that (1) high inositol polyphosphates may represent a link between IP3 and cAMP pathways; (2) the IP3-induced increase of MEPP amplitude is likely to be due to its high inositol metabolites; (3) PI 3-K is not involved in the IP4 and IP6-mediated MEPP frequency increases, but may be involved in MEPP size.