D-serine released by astrocytes in brainstem regulates breathing response to CO2 levels.

Central chemoreception is essential for adjusting breathing to physiological demands, and for maintaining CO2 and pH homeostasis in the brain. CO2-induced ATP release from brainstem astrocytes stimulates breathing. NMDA receptor (NMDAR) antagonism reduces the CO2-induced hyperventilation by unknown mechanisms. Here we show that astrocytes in the mouse caudal medullary brainstem can synthesize, store, and release D-serine, an agonist for the glycine-binding site of the NMDAR, in response to elevated CO2 levels. We show that systemic and raphe nucleus D-serine administration to awake, unrestrained mice increases the respiratory frequency. Application of D-serine to brainstem slices also increases respiratory frequency, which was prevented by NMDAR blockade. Inhibition of D-serine synthesis, enzymatic degradation of D-serine, or the sodium fluoroacetate-induced impairment of astrocyte functions decrease the basal respiratory frequency and the CO2-induced respiratory response in vivo and in vitro. Our findings suggest that astrocytic release of D-serine may account for the glutamatergic contribution to central chemoreception.Astrocytes are involved in chemoreception in brainstem areas that regulate breathing rhythm, and astrocytes are known to release D-serine. Here the authors show that astrocyte release of D-serine contributes to CO2 sensing and breathing in brainstem slices, and in vivo in awake unrestrained mice.

Development and pH sensitivity of the respiratory rhythm of fetal mice in vitro.

In newborn and adult mammals, chemosensory drive exerted by CO(2) and H(+) provides an essential tonic input: without it the rhythm of respiration is abolished. It is not known, however, whether this chemosensory drive and the respiratory rhythm appear simultaneously during development. In isolated brainstem-spinal cord preparations from fetal mice, we determined at what stage of fetal life the respiratory rhythm appeared in third to fifth cervical ventral roots (phrenic motoneurons) and whether this fetal rhythm was sensitive to chemosensory inputs. A respiratory-like rhythm consisting of short duration bursts of discharges recurring at 2-16 min(-1) was detected in two of nine embryonic day 13 fetuses; it was abolished by transection of the spinal cord between the first to second cervical segments and was phase-related to rhythmic activity from medullary units of the ventral respiratory group. At embryonic day 13, it coexisted with a slow rhythm (0.1-2.0 min(-1)) of long duration bursts of action potentials which was generated by the spinal cord. At later fetal stages, the respiratory-like rhythm became more robust and of higher frequency, while the spinal cord rhythm became less obvious. At all fetal stages, acidification of the superfusion medium from pH 7.5-7.2 or 7.4-7.3 or 7.4 to 7.2 increased the frequency of both the respiratory-like and the spinal cord rhythms. In addition, acidification reduced the amplitude of the integrated burst activity of the spinal cord rhythm of embryonic day 13-embryonic day 16 fetuses and the respiratory-like rhythm of embryonic day 17 and older fetuses. Our results indicate that the rhythms transmitted by phrenic motoneurons during fetal development are chemosensitive from early fetal stages. Through its effects on induction and patterning of the rhythm, chemosensory drive may play a role in activity-dependent formation of respiratory neural networks.

In vitro approach to the chemical drive of breathing.

Since its introduction two decades ago, the isolated brain stem-spinal cord preparation of neonatal rodents has been the preferred method used to reveal the mystery underlying the genesis of the respiratory rhythm. Little research using this in vitro approach has focused on the study of the central respiratory chemosensitivity. Some unexpected findings obtained with the brain stem-spinal cord preparation have added new questions that challenge our previous theoretic framework. Some of these findings are addressed here.

Allopregnanolone-induced modification of presynaptic basal and K+-induced [3H]-norepinephrine efflux from rat cortical slices during the estrous cycle.

Superfused frontal slices of cerebral cortex were preloaded with [3H]-norepinephrine ([3H]NE). Basal [3H]NE efflux and K+-induced [3H]NE release were studied during the estrous cycle and in the presence of neurosteroids. Basal [3H]NE efflux showed estrous cycle-related variations, with lowest values found during estrus and diestrus II. Allopregnanolone (10(-9) M) potentiated basal [3H]NE efflux from the 1st minute of its application; the effect of the steroid was still present after 20 min. This effect was also dependent upon the estrous cycle, since basal [3H]NE efflux was mainly increased during estrus diestrus I, and to a lesser degree only during proestrus. During diestrus II and after ovariectomy, basal [3H]NE efflux was no longer affected by the neurosteroid. In the presence of yohimbine (10(-6) M), the effect of allopregnanolone on basal efflux was potentiated only during the first 3 min but vanished thereafter. Allopregnanolone (10(-9) M) potentiated the K+-induced [3H]NE release during estrus, but pregnenolone (10(-9) M) was ineffective, suggesting specificity of the neurosteroid. Yohimbine (10(-6) M) also potentiated K+-induced [3H]NE release. When applied simultaneously with allopregnanolone (10(-9) M), a potentiating effect on [3H]NE release was observed. The present results suggest that allopregnanolone is a neurosteroid able to modulate norepinephrine release in the cerebral cortex in an estrous cycle-dependent manner, and that the effect could involve noradrenergic alpha-2 receptors.

Noradrenergic neurons release both noradrenaline and neuropeptide Y from a single pool: the large dense cored vesicles.

In peripheral adrenergic nerve endings, noradrenaline is stored in two different types of vesicles, the large and the small dense cored vesicles. A systematic study was undertaken to examine the release of noradrenaline and neuropeptide Y from dog spleen and rat vas deferens under various conditions of stimulation, particularly those which previously have demonstrated a differential regulation of exocytosis of the different types of storage vesicles. Here we present evidence that noradrenaline is released by exocytosis exclusively from the large dense cored vesicles, in which it is stored together with neuropeptide Y. Upon a single stimulation (at frequencies varying from 2-20 Hz), the release of noradrenaline and neuropeptide Y from the dog splenic nerve increased with the frequency of stimulation, but the ratio of noradrenaline to neuropeptide Y remained constant. After repeated stimulation of the splenic nerve, both substances' overflow decreased gradually and in parallel to values of 12.5% and 11.1% of the first stimulation for noradrenaline and neuropeptide Y, respectively. Similarly, repeated stimulation of the rat vas deferens (of which only 2-10% is large dense cored vesicles, whereas in the dog splenic nerve the large dense cored vesicles make up 30-40% of the total vesicle population) with 120 mM K+, in the presence of phentolamine, caused a gradual and parallel decline in the release of noradrenaline and neuropeptide Y (31.6% and 34.0%, respectively). Moreover, omega-conotoxin (10(-8) M to 10(-5) M) had a similar inhibitory effect on the release of both substances, alpha-latrotoxin (10(-9) M) evoked a parallel release of both noradrenaline and neuropeptide Y. The results indicate that noradrenaline in peripheral noradrenergic nerves is released exclusively from large dense cored vesicles by an exocytotic mechanism.

Synaptic like microvesicles: do they participate in regulated exocytosis?

Synaptic like microvesicles (SLMV) form a pleiomorphic population of vesicles present in neuroendocrine cells of the pituitary and the adrenal medulla. Their composition and biogenesis is very similar to small synaptic vesicles (SSV) present in nerve terminals. Membrane proteins of SSV involved in fusion (synaptotagmin) and trafficking (Rab 3) together with synaptophysin have been identified on SLMV. The SLMV from pancreatic beta cells store GABA, those present in chromaffin cells catecholamines and/or acetylcholine. We conclude that SLMV may participate in regulated exocytosis based on (a) their localization near release sites, (b) their content and (c) their membrane composition. The functional meaning and the way this process is regulated remain to be elucidated.

Subcellular localization of synaptophysin in noradrenergic nerve terminals: a biochemical and morphological study.

The subcellular localization of synaptophysin was investigated in noradrenergic nerve terminals of bovine vas deferens and dog spleen and compared with membrane-bound and soluble markers of noradrenergic storage vesicles. At the light microscopical level chromogranin A- and cytochrome b561-immunoreactivity revealed an identical and very dense innervation of the entire vas deferens. In the case of synaptophysin, most immunoreactivity was found only in the outmost varicosities closest to the lumen, which were also positive for chromogranin A. Small dense-core vesicles of dog spleen were purified using a combination of velocity gradient centrifugation and size exclusion chromatography. Small dense-core vesicles were enriched 64 times as measured by the noradrenaline content. Enrichments for dopamine-beta-hydroxylase were in a similar range. Synaptophysin-containing vesicles were smaller in size and they did not contain the typical noradrenergic markers dopamine-beta-hydroxylase, cytochrome b561, and noradrenaline. Instead, they might store adenosine triphosphate (ATP). A greater part of synaptophysin immunoreactivity was consistently found at high sucrose densities at the position of large dense-core vesicles. We conclude that in the noradrenergic nerve terminal: (1) small dense-core vesicles have a membrane composition similar to large dense-core vesicles, indicating that the former are derived from the latter, and (2) synaptophysin seems not to be present on small dense-core vesicles. We suggest the possibility that synaptophysin-containing vesicles form a residual population whose role in neurotransmission has been taken over by large and small dense-core vesicles following noradrenergic differentiation.

Co-storage in large 'dense-core' vesicles of dopamine and cholecystokinin in rat striatum.

The subcellular localization of cholecystokinin in the striatum--an area where a high density of cholecystokinin containing terminals has been demonstrated--was studied using biochemical techniques. Cholecystokinin containing vesicles were partially purified using iso-osmotic Ficoll gradients. As judged from their size and their buoyant density in isopycnic gradients, cholecystokinin containing vesicles represent large 'dense-core' vesicles. Negative staining and subsequent immunolabelling for synaptophysin at the electron microscopical level, showed labelled vesicles of 50-70 nm. binding of dihydrotetrabenazine was detected in the cholecystokinin containing fractions. The results suggest that dopamine is co-stored with cholecystokinin in large dense vesicles in rat striatum.

Receptor-mediated internalization of angiotensin II in bovine adrenal medullary chromaffin cells in primary culture.

Binding and internalization of angiotensin II (AII) were studied on bovine adrenal medullary cells in primary culture. Binding of [125I]AII was reversible, saturable, specific and showed high affinity. AII was found to be internalized by bovine adrenal medullary cells. Monensin increased whereas phenylarsine oxide (PhAsO) decreased the internalization. Excess of unlabelled AII or saralasin could block the internalization, indicating a receptor mediated internalization process. The kinetic analysis indicated that, during the first 4 min, about 25% of the membrane bound ligand was internalized per min and the recycling of internalized ligand and receptor initiated around 4 min.

Differences in the distribution of cytochrome b561 and synaptophysin in dog splenic nerve: a biochemical and immunocytochemical study.

Compared with neurons of the CNS, the organization of the peripheral adrenergic axon and nerve terminal is more complex because two types of neurotransmitter-containing vesicles, i.e., large (LDVs) and small dense-core vesicles, coexist with the axonal reticulum (AR) and the well-characterized small synaptic vesicles. The AR, which is still poorly examined, is assumed to play some role in neurosecretion. We have studied the subcellular localization of noradrenaline, cytochrome b561, and synaptophysin in control and ligated dog splenic nerve using both biochemical and ultrastructural approaches. Noradrenaline and cytochrome b561 coaccumulated proximal to a ligation, whereas distally only the latter was found. Despite a codistribution with noradrenaline at high densities in sucrose gradients, synaptophysin did not accumulate on either side of the ligation. At the ultrastructural level, cytochrome b561 immunoreactivity was found on LDVs and AR elements, both accumulating proximal to the ligation. Distally, the multivesicular bodies (MVBs), immunolabeled for cytochrome b561, account for the retrograde transport of LDVs and AR membranes retrieved at the nerve terminal. No synaptophysin immunoreactivity could be detected on LDVs, AR, or MVBs. The results obtained from the ligation experiments together with the ultrastructural data clearly illustrate that synaptophysin is absent from LDVs and AR elements in adrenergic axons.

Catecholamines are present in a synaptic-like microvesicle-enriched fraction from bovine adrenal medulla.

"Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS, a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation, we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine beta-hydroxylase and cytochrome b561. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [3H]Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin; however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of approximately 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine beta-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b561 was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.

Simultaneous purification of the neuroproteins synapsin I and synaptophysin.

A procedure for the simultaneous purification of synapsin I and synaptophysin from calf brain was developed. Demyelinated membranes were extracted with 2% Triton X-100 and 2 M KCl. The extracted proteins were separated by weak cation-exchange chromatography on carboxymethyl-Sepharose Fast Flow. Synaptophysin was finally purified by preparative sodium dodecyl sulphate-polyacrylamine gel electrophoresis and synapsin I by affinity chromatography using a calmodulin-Sepharose column. The recovery obtained was 40 micrograms/g in brain for synaptophysin and 25 micrograms/g in brain for synapsin I.

Biochemical characterization of particles from rat striatum containing dopamine, cholecystokinin or both.

Cholecystokinin (CCK) containing particles were isolated from rat striatum. After differential and isopycnic sucrose density gradient centrifugation, CCK containing particles equilibrated around 1.2 M sucrose. Dopamine (DA) containing particles equilibrated around the same buoyant density, but the colocalization of a cytosolic as well as a mitochondrial enzyme marker and the low enrichment at this density indicated an incomplete isolation of CCK containing particles from other subcellular organelles. Alternatively, differential centrifugation and gel filtration on Sephacryl S-1000 did yield advanced isolation of CCK containing vesicles. These vesicles also contain particle-bound DA. During hypo-osmotic lysis, these CCK and/or DA containing particles behaved like synaptic vesicles. Therefore, it was concluded that CCK containing vesicles from rat corpus striatum were indeed isolated and characterized and that the same or similar vesicles contain DA as well as CCK.

Bradykinin modulates the release of noradrenaline from vas deferens nerve terminals.

To asses whether bradykinin influences the release of noradrenaline from the adrenergic varicosities of the vas deferens, tissues were loaded with 3H-noradrenaline. Upon electrical depolarization bradykinin increased in a concentration-dependent fashion, the overflow of tritium from the mouse or rat vas deferens. The 3H-overflow is dependent on the external Ca2+concentration suggesting neuronal release of 3H-noradrenaline. The present results add evidence to the hypothesis that bradykinin modulates the release of noradrenaline from peripheral sympathetic nerve terminals via the activation of a presynaptic mechanism.

Identification of pre- and postsynaptic bradykinin receptor sites in the vas deferens: evidence for different structural prerequisites.

The effect of bradykinin on the neuroeffector junction of the isolated rat vas deferens was studied in tissues stimulated transmurally at a frequency of 0.15 Hz. Bradykinin caused two distinct and independent actions: it potentiated the magnitude of the muscular response to the electrically driven twitches and, in addition, contracted the smooth muscle generating an increased muscular tone. The former action is referred to as the neurogenic or presynaptic effect, whereas the latter effect is called the musculotropic or postjunctional action. The neurogenic effect was abolished by tetrodotoxin or tissue denervation either by cold storage or chemical sympathectomy after 6-hydroxydopamine administration. However, these procedures did not significantly modify the musculotropic potency of bradykinin. Both actions of the peptide are receptor-mediated, as minor structural modifications in the amino acid sequence caused significant changes in biological potency. In addition, the peptide analog, [Thi5,8-D-Phe7]-bradykinin, behaved as an agonist at the presynaptic site but as an antagonist at the muscular site. The most potent peptide analog to produce the neurogenic effect was Met-Lys-bradykinin followed by Lys-bradykinin and [Tyr8]-bradykinin. In contrast, the potency of these peptide analogs acting at the postsynaptic site was about the same. des Arg9 bradykinin and des Arg9-[Leu8]-bradykinin were inactive at the pre- and postjunctional site. The neurogenic action of bradykinin was not mimicked by angiotensin II, neurotensin, substance P or vasopressin.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin-acetylcholinesterase interaction: Specific detachment of class I-A forms and binding of class I and II-A forms to heparin-agarose.

This study describes the specificity, time-course and characteristics of the solubilization of class I-A forms of AChE by heparin, from the endplate regions of rat diaphragm muscle. Heparin fractions which differed in size charge, anticoagulant activity and capacity to bind type I collagen, were probed in their ability to extract AChE. No differences were found among all the fractions tested. Affinity chromatography on heparin-agarose of class I- and class II-A forms of esterase showed that both classes were able to bind to the column with the same relative affinity. Our results establish the use of heparin, as a solubilizing agent for the class I-A. The existence of a heparin-binding domain in class I- and class II-A forms of AChE, opens the possibility, that heparan sulfate proteoglycans could be involved in the anchorage of both types of esterase to synaptic regions. Finally, our results suggest that class I and class II-A do not correspond to intrinsically distinct molecules, but rather to identical molecules engaged in different interactions in the tissue.