The storage of endogenous noradrenaline in sympathetic nerve terminals

1. The subcellular distribution of noradrenaline in sympathetic nerve terminals of rat vas deferens and cat spleen has been studied by cell fractionation methods combined with fluorescence and electronmicroscopic histochemical methods for noradrenaline.2. Pinched-off axon varicosities (synaptosomes) were isolated and identified by fluorescence and electronmicroscopy in the mitochondrial pellet.3. The proportion of large to small dense-cored vesicles in electronmicrographs of sympathetic nerve terminals varies in different organs. In rat vas deferens 4% and in cat spleen 20% are large vesicles.4. Density gradients of rat vas deferens have a single low density peak of noradrenaline at 0.6 M sucrose, whereas those of cat spleen have an additional peak of noradrenaline at 1.1 M sucrose.5. Small dense-cored vesicles were identified electronmicroscopically in the low density fractions and large dense-cored vesicles in the high density fractions from density gradients.6. We conclude that both small and large dense-cored vesicles store noradrenaline.     

Noradrenaline content correlated to matrix density in small noradrenergic vesicles from rat seminal ducts

The relation between matrix density and noradrenaline content has been investigated in a fraction of small noradrenergic storage vesicles obtained from seminal ducts of castrated rats by density gradient centrifugation. This preparation contained numerous dense-cored small vesicles with an increased diameter but with a similar appearance to those in nerve terminals in the intact vas deferens.After incubation of the vesicle fraction for 20 min at 30°C in the presence of noradrenaline, Mg²⁺ and adenosine 5'-triphosphate, about 50–60% of the vesicles with a diameter of 40–70 nm contained dense cores, in some regions of the sediment even up to 80%. This correlated well with a high level of sedimentable noradrenaline, 150% of control.Incubation of vesicles without exogenous noradrenaline in the presence of Mg²⁺ and adenosine 5'-triphosphate yielded mostly electron-lucent vesicles, and biochemically only 40% of the control level of noradrenaline was retained in the particulate fraction after centrifugation. A similar depletion of electron dense-core material was observed after incubation with tyramine, Mg²⁺ and adenosine 5'-triphosphate, where only 55% of the sedimentable noradrenaline as compared to control was retained. In this respect the small dense-cored vesicles differed markedly from the large dense-cored vesicles purified from bovine splenic nerve, where the dense cores were retained in spite of a 70–80% depletion of noradrenaline after incubation in the presence of tyramine.Incubation with 5-hydroxydopamine did not significantly increase the electron density of the small dense-cored vesicles, in contrast to the marked effect of this agent when administered in vivo. Incubation with reserpine and atractyloside did not affect either noradrenaline release or vesicle morphology significantly, whereas treatment with N-ethylmaleimide gave a 60% decrease in particulate noradrenaline content but only a moderate reduction in electron density.The results indicate that both noradrenaline and, to a lesser extent, other matrix material in the presence of Mg²⁺ and adenosine 5′-triphosphate are capable of forming electron-dense cores, and that noradrenaline is more important for dense-core formation in small noradrenergic vesicles than it is in the large vesicles.     

Parallel changes in ultrastructure and noradrenaline content of nerve terminals in rat vas deferens following transmitter release

Transmural electrical stimulation and exposure to incubation media where some or all of the Na⁺ had been replaced with K⁺ were used to elicit transmitter release. Changes in noradrenaline content and ultrastructure of the nerve terminal varicosities in rat vas deferens were measured. Electrical stimulation in the presence of 4-aminopyridine had little effect, but high [K⁺] solutions caused a parallel reduction in noradrenaline content and the number of small dense-cored vesicles; large densecored vesicles showed no change, and small clear vesicles increased in number. In spite of a reduction in total vesicle number there was no evidence of expansion of the varicosity membrane.The parallel fall in noradrenaline content and in the number of small dense-cored vesicles suggests that the latter are the source of the released noradrenaline under the conditions of high [K⁺] stimulation we have used.     

Biochemical and functional properties of large and small dense-core vesicles in sympathetic nerves of rat and ox vas deferens

A comparative study of the noradrenaline storing vesicles in vas deferons from ox and rat was performed. Microsomal fractions were subjected to density gradient centrifugation. In rat, noradrenaline and dopamine β-hydroxylase were mainly present in the upper fractions of the gradient, which is consistent with the predominance of light (small dense-core) vesicles in this species. In ox, noradrenaline, dopamine and dopamine β-hydroxylase were found in the gradient in a bimodal distribution. This is consistent with the presence of about equal numbers of small and large dense-core vesicles in this species. On the other hand, chromogranin A, immunologically related proteins and enkephalin-like immunoreactivity were only present in the dense (large dense-core) vesicle population. In order to study the capability of light and dense vesicles to synthesize noradrenaline we “pulse-labelled” ox vasa deferentia with [³H]tyrosine. Already 3.5 min after the pulse both types of vesicles contained [³H]noradrenaline and [³H]dopamine. During longer “chase” periods the amount of [³H]dopamine gradually declined.We conclude that dense (large dense-core) vesicles contain chromogranin A, immunologically related proteins and enkephalin-like immunoreactivity whereas light (small dense-core) vesicles are devoid of these components. Both types of vesicles contain dopamine β-hydroxylase and can synthesize noradrenaline from dopamine under in vivo conditions.     

Developmentally regulated changes of glutamate binding sites in mouse deep cerebellar nuclei

The expression of L-[³H]glutamate binding sites of different ionic and pharmacological sensitivities was studied in mouse deep cerebellar nuclei during early postnatal development by means of in vitro autoradiography. Ca²⁺/Cl⁻-dependent, quisqualate/AMPA/ibotenate-sensitive, and APB-insensitive binding sites are present at high density in the deep cerebellar nuclei of young animals, but greatly decrease between the 10th and 25th postnatal day and remain low in the adult. The density of Ca²⁺/Cl⁻-independent binding sites remains low and constant during the whole of postnatal development. The possible involvement of the Ca²⁺/Cl⁻-dependent binding sites in brain development is discussed.     

Endogenous protein phosphorylation in chick and rat brain synaptic membranes

The protein kinase activities endogenous to synaptic membranes prepared by an identical procedure from avian (chick) and mammalian (rat) brains were compared. Both species showed similar responses towards both protein kinase effector molecules cyclic adenosine monophosphate and Ca²⁺. K_app for cyclic adenosine monophosphate-dependent protein kinase activity occurred at 0.4–0.8μM cAMP and K_app for Ca²⁺-dependent, calmodulin-requiring protein kinase activity occurred at 1–2μM Ca²⁺ (free ion concentration) both in the absence or presence of calmodulin added to the reaction mixture. This suggests that endogenous calmodulin in these membranes was able to modulate the Ca²⁺-dependent, calmodulin requiring protein kinase activity. After EGTA-treatment of the membranes to remove endogenous Ca²⁺ and calmodulin, no significant response towards Ca²⁺ on the phosphorylation of the membrane polypeptides was measured unless exogenous calmodulin was added after which the K_app for Ca²⁺ was increased to 15μM Ca²⁺ (free ion concentration). There was a difference in the maximal levels of kinase activity in these membranes with chick membranes containing 57% less cyclic adenosine monophosphate-dependent protein kinase activity, but 65% more Ca²⁺-dependent, calmodulin-requiring protein kinase activity than the rat membranes. Similar results were determined when either low (5 μM) or high (5.8mM) concentrations of adenosine 5′-triphosphate were added to the reaction mixtures.

Besides certain species differences in the molecular weights of the resulting phosphoproteins, we observed several major differences with respect to the absence or presence of some of the phosphoproteins. Chick synaptic membranes may lack the cyclic adenosine monophosphate-requiring, microtubule-associated phosphoprotein, MAP₂, one of the 2 neurospecific, cyclic adenosine monophosphate-requiring and Ca²⁺, calmodulin-requiring phosphoproteins (Protein Ib, although Protein Ia apparently is present), and the Ca²⁺-requiring, calmodulin-independent, ACTH-sensitive phosphoprotein, B50.

The phenothiazines, trifluoperazine, fluphenazine and chlorpromazine were found to inhibit the Ca²⁺-dependent, calmodulin-requiring protein kinase activities of both the chick and rat synaptic membranes. This inhibition appeared to be specific for calmodulin because at the same concentrations the phenothiazine analogue, chlorpromazine-sulfoxide, had no effect on this activity. Also found to inhibit Ca²⁺-dependent calmodulin-requiring protein kinase activity were dibucaine and adrenocorticotropin.

These data suggest that rat forebrain synaptic plasma membranes are activated by cyclic adenosine monophosphate to a greater extent than are chick forebrain synaptic plasma membranes while the chick membranes are activated to a greater extent by Ca²⁺, calmodulin than are the rat membranes, though similarities dominate comparison of the two membrane systems.     

Effect of Nerve Stimulation In Vitro on the Noradrenaline Content of the Rat Vas Deferens in the Presence of Inhibitors of Noradrenaline Uptake and Synthesis

Isolated rat vas deferens preparations were intermittently field stimulated (30 s every min) for 2 or 4 h at 7 or 25 imp/s. At the low stimulation frequency only a very small reduction (7 %) of the endogenous noradrenaline (NA) stores was seen during 4 h stimulation while it was about 15 % after 2 h and 37 % after 4 h at 25 imp/s. Addition of drugs known to inhibit NA synthesis (a-methyl-p-tyrosine, a-MPT, 4times10^-4M) or uptake (cocaine, 10^-5M) did not apparently influence neither the spontaneous nor *the nerve-induced reduction of the endogenous NA stores. The present results seem to support the hypothesis that the functional transmitter pool of the short adrenergic neurons of the vas deferens is very small, and that the residual NA reuptake and synthesis after pharmacological blockade, together with minute refilling from the large storage pools, are able to maintain transmitter homeostasis without significant changes of the total endogenous NA content of the vas deferens.     

Asymmetric distribution of purinergic and adrenergic neurotransmission cooperates in the motor activity along the rat vas deferens

The distribution of purinergic and adrenergic responses in the epididymal and prostatic segment of the rat vas deferens were studied in vitro. Prazosin antagonizes the twitch elicited by electrical stimulation mainly in the epididymal segment while alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-mATP) preferentially inhibits the response of the prostatic segment. Using both prazosin plus alpha,beta-mATP, the response to field stimulation was completely inhibited. Concentration-response curves revealed that adrenergic compounds elicited a greater contraction in the epididymal portion than in the prostatic end of the ductus. Purinergic compounds caused a contraction of greater magnitude in the prostatic portion. The results suggest that adrenergic and purinergic mechanisms are asymmetrically distributed along the vas deferens reflecting a gradient of adrenergic and purinergic receptors along the ductus.     

Further evidence for adrenergic transmission in the human vas deferens.

1. Isolated portions of human vas deferens responded to field stimulation of the intramural nerve fibres or to exogenously applied noradrenaline with rhythmical contractions of both longitudinal and circular muscle layers. 2. In guinea-pig and rabbit vas deferens field stimulation produced an initial rapid 'twitch' response which was not found with human vasa. 3. Responses of the human vas to field stimulation were depressed by the alpha-adrenoceptor blocking agents phentolamine and yohimbine. 4. It is concluded that the motor innervation of the human vas deferens is adrenergic and the relevance of this to the physiological operation of the tissue is discussed.     

Sympathetic axons and nerve terminals: the protein composition of small and large dense-core and of a third type of vesicles

Homogenates of bovine splenic nerve and of vas deferens were subjected to differential and density gradient centrifugation to investigate their noradrenaline-storing organelles. The subcellular fractions obtained were analysed by immunoblotting in order to define the presence of various antigens in small dense-core and large dense-core vesicles. In both large granule and microsomal fractions from splenic nerve only one type of noradrenaline-storing vesicle was found, which represents the large dense-core vesicles. These organelles contained chromogranin A, chromogranin B, cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, dopamine beta-hydroxylase and the monoamine carrier which are also present in adrenal chromaffin granules. The subcellular distribution of synaptin/synatophysin was more complex since this protein was apparently present in two organelles: in a light vesicle which did not contain significant amounts of antigens found in large dense-core vesicles (dopamine beta-hydroxylase, cytochrome b-561 and the monoamine carrier) and in the dense fractions of the gradient, possibly within large dense-core vesicles. In the microsomal gradient from vas deferens several markers (catecholamines, synaptin/synaptophysin and dopamine beta-hydroxylase) were found in a bimodal distribution, which is consistent with their presence in small and large dense-core vesicles. When the larger granules were removed with higher centrifugation speed a microsomal fraction containing only light vesicles was obtained. After gradient centrifugation of this fraction several components (catecholamines, dopamine beta-hydroxylase, cytochrome b-561, the monoamine carrier and synaptin/synaptophysin) were concentrated in a peak at low density; apparently only small dense-core vesicles were now present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of neuroeffector transmission in guinea-pig pulmonary artery and vas deferens by exogenous nitric oxide

Cederqvist , B. & Gustafsson , L. E. 1994. Modulation of neuroeffector transmission in guinea-pig pulmonary artery and vas deferens by exogenous nitric oxide. Acta Physiol Scand 150, 75–81. Received 11 March 1993, accepted 30 July 1993. ISSN 0001–6772. Department of Physiology and Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden. Blockade of nitric oxide (NO) synthesis enhances contractile responses to transmural nerve stimulation in guinea-pig pulmonary artery, indicating neuromodulation by endogenous nitric oxide. In the present study, neuromodulatory effects of exogenous NO were examined in guinea-pig pulmonary artery and vas deferens. Application of NO as acid nitrite, to the guinea-pig pulmonary artery, inhibited the contractile response to transmural nerve stimulation as well as contractions to exogenous noradrenaline. The inhibition occurred in a dose-dependent manner. Acid nitrite did not affect stimulation-induced release of [³H]noradrenaline in guinea-pig pulmonary artery. In the guinea-pig vas deferens, contractile ‘twitch’ responses to brief (25 pulses) transmural nerve stimulation and stimulation-induced release of [³H]noradrenaline was unaffected by acid nitrite. However, ‘twitch’ responses to prolonged stimulation were inhibited by acid nitrite, and tonic ‘hump’ responses were either enhanced or unaffected by acid nitrite. In conclusion, exogenous nitric oxide exerted an inhibition of adrenergic neurotransmission post-junctionally in guinea-pig pulmonary artery. In guinea-pig vas deferens, exogenous nitric oxide affected adrenergic and/or non-adrenergic non-cholinergic neurotransmission in a complex fashion, however without alteration in noradrenaline release. The data support a role for NO in modulation of neuroeffector transmission, especially by modulation of effector sensitivity, whereas modulation of noradrenaline release seems an unlikely role for NO in these and similar peripheral tissues.     

Sympathetic denervation of the smooth muscle of the vas deferens

1. The post-ganglionic nerve fibres to the vas deferens of the guinea-pig and rat were interrupted in vivo by stripping one vas deferens of its serous coat; the other vas deferens was left intact as a control.2. Four to eight days later the stripped vas deferens did not contract in response to electrical transmural stimulation in vitro at 0.1 msec pulse duration. Pulses of 1.0 msec duration produced small contractions which were not abolished by local anaesthetic or adrenergic neurone-blocking drugs.3. Log dose-response curves to noradrenaline were, for stripped vasa deferentia, to the left of those for control vasa. The increase in sensitivity to noradrenaline at 8 days was about sixteenfold for rat vasa and about tenfold for guinea-pig vasa. Tyramine did not contract stripped vasa from guinea-pigs or rats.4. The noradrenaline and adrenaline content of guinea-pig and rat vasa was greatly reduced or abolished 8 days after the stripping operation.5. Fluorescent nerve terminals were usually absent when transverse sections of stripped vasa were examined by fluorescence microscopy after treatment by the formaldehyde condensation method for demonstrating catecholamines.6. It is concluded that post-ganglionic sympathetic denervation is achieved by stripping the vas deferens in vivo of its serous coat and mesenteric attachments.     

Adrenergic component in the mechanism of action of sodium hydroxybutyrate

Using the isolated rat vas deferens as the model the effects of sodium hydroxybutyrate on reserves of adrenergic mediator in nerve fibers and synaptic vesicles and the uptake and accumulation of exogenous noradrenalin were studied. It was shown by spectrofluorometric, fluorescence-histochemical, and cytochemical electronmicroscopic methods that sodium hydroxybutyrate does not affect the reserves of adrenergic mediator but can block the uptake and accumulation of exogenous noradrenalin.     

Differences in Secretory Excitability between Short and Long Adrenergic Neurons: Comparison of 3H-Noradrenaline Secretion Evoked by Field Stimulation of Guinea-Pig Vas Deferens and Human Blood Vessels

On comparing the secretion of noradrenaline (NA) evoked by electrical field stimulation of the ‘short’ adrenergic nerves of guinea-pig isolated vas deferens (Sjöstrand 1962) with that evoked by similar stimulation of the ‘long’ adrenergic nerves of human isolated omental blood vessels, using identical experimental conditions, it was found that the two types of nerve differ markedly in ‘secretory excitability’. While it was possible to adjust the strength and/or duration of stimulus shocks to a level causing a well-defined maximum in ³H-NA output from the human vasomotor nerves, no such ‘supramaximal’ level could be reached in the nerves of guinea-pig vas deferens.     

Ion channels and the control of swimming in the Xenopus embryo

The Xenopus embryo has been well studied and the circuitry underlying motor pattern generation largely elucidated. We have extended this analysis by determining the roles of individual voltage- and ligand-gated ion channels in controlling the motor pattern for swimming and two mechanisms that control rundown of this pattern.

Xenopus embryo spinal neurons possess at least six classes of ion channel: a fast Na⁺ channel; a mixture of kinetically similar Ca²⁺ channels; a fast K⁺ channels; a slow K⁺ channel; a Na⁺-dependent K⁺ channel; and a slowly activating Ca²⁺-dependent K⁺ channel.

The roles of the voltage-gated currents in determining neuronal firing properties and operation of the locomotor circuitry have been examined both pharmacologically and in realistic computer simulations.

Model neurons fire repetitively in response to current injection. The Ca²⁺ current seems essential for repetitive firing. The fast K⁺ current appears mainly to control spike width, whereas the slow K⁺ current exerts a powerful influence on repetitive firing. These predictions from the model have been confirmed by the use of specific pharmacological blockers of the fast and slow K⁺ currents.

Both the model network and the real spinal locomotor circuit appear to tolerate a wide variation in the relative strengths of the component synapses but are very sensitive to the magnitudes of the voltage-gated currents. In particular the slow K⁺ current, despite being a small component of the total outward current, plays a critical role in stabilizing the motor pattern.

Like many other rhythmic motor patterns, swimming in the Xenopus embryo is episodic; it undergoes run-down and self-termination even in the absence of sensory inputs. The slow Ca²⁺-dependent K⁺ current appears to play a role in the self-termination of swimming. However, intrinscic modulation mediated by the release of ATP and production of adenosine in the extracellular space appears to be a very powerful determinant of run-down of the motor pattern.     

Subcellular distribution of adenylate cyclase and cyclic adenosine monophosphate phosphodiesterase in rat and guinea-pig vas deferens

Cyclic adenosine 3′:5′-monophosphate (cyclic AMP) is assumed to play a role in catecholamine synthesis and release. In order to determine if cyclic AMP metabolism at the level of the storage granule is important in this respect the subcellular distribution of adenylate cyclase and cyclic AMP phosphodiesterase was determined in vas deferens from normal rats, castrated rats and castrated guinea-pigs. The phosphodiesterase activity was mainly found in the soluble fractions, while the adenylate cyclase was associated with sedimentable material. When vas deferens homogenates from both intact and castrated rats were subjected to sucrose density gradient centrifugation the main part of the adenylate cyclase activity was found associated with membrane fragments at 0.5–0.6 m sucrose. The distribution of adenylate cyclase activity in the density gradient parallelled that of 5′-nucleotidase, but was different from that of noradrenaline. Also in the guinea-pig, adenylate cyclase tended to have a different distribution from that of noradrenaline.The adenylate cyclase activity in, all fractions was stimulated by fluoride and guanosine triphosphate. Noradrenaline, prostaglandin E₂, 2-chloroadenosine and phenylisopropyladenosine stimulated adenyl cyclase activity in nuclear and mitochondrial fractions, but only to a small extent, if at all, in the fractions collected from the density gradient.The results do not indicate that adenylate cyclase activity in vas deferens homogenates is associated with catecholamine storage vesicles. Hence, cyclic AMP metabolism at the level of the storage granule is probably not involved in transmitter turnover.     

Distribution and intraneuronal trafficking of a novel member of the chromogranin family, NESP55, in the rat peripheral nervous system

NESP55 (neuroendocrine secretory protein of M_r 55 000) is a novel member of the chromogranin family. In the present study, we have investigated the distribution, axonal transport and proteolytic processing of NESP55 in the peripheral nervous system. The amount of NESP55 immunoreactivity in adrenal gland was more than 240 times higher than that in the vas deferens. Double or triple immunostaining demonstrated that NESP55 immunoreactivity was highly co-localized with tyrosine hydroxylase immunoreactivity in bundles of thin axons and postganglionic sympathetic neurons; that NESP55 immunoreactivity also co-existed with vesicular acetylcholine transporter immunoreactivity in large-sized axons in sciatic nerves, and that NESP55 immunoreactivity overlapped with calcitonin gene-related peptide immunoreactivity in some large-sized axons, but NESP55 immunoreactivity was not detected in sensory neurons. Strong NESP55 immunoreactivity was found in cell bodies and axons, but it was not detectable in any terminal region by immunohistochemistry. In crush-operated sciatic nerves, NESP55 immunoreactivity could be found as early as 1 h after operation, and accumulated amounts increased substantially with time. However, NESP55 immunoreactivity was only observed in axons proximal to the crush, but none or very little distal to the crush, which was consistent with the data from radioimmunoassay. Finally, extracts of the normal and crushed sciatic nerve and vas deferens were subjected to high-performance liquid chromatography followed by radioimmunoassay. The results indicate that NESP55 is processed slowly to small peptides (GAIPIRRH) during axonal transport. NESP55 immunoreactivity was only detected in axons proximal to the crush.

The data in the present study indicate that NESP55 immunoreactivity is widely distributed in adrenergic, cholinergic, and peptidergic neurons, but not in sensory neurons, and that this peptide is anterogradely, but not retrogradely, transported with fast axonal transport and slowly processed to smaller peptides during axonal transport in the peripheral nervous system.     

Regional variations in endoplasmic reticulum in the vas deferens of normal and vasectomized rats

The fine structure of the epithelium of different parts of the rat vas deferens was studied in normal rats and at intervals of up to nine months after vasectomy. The cytology of the columnar epithelial cells showed regional variations particularly in the type and extent of endoplasmic reticulum. Cells of the proximal part of the vas deferens were characterized by basal and perinuclear granular endoplasmic reticulum, a large Golgi apparatus, and numerous apical microvilli, vesicles, and vacuoles. In contrast, the columnar cells of the distal portion of the vas deferens contained large amounts of smooth endoplasmic reticulum, much of which assumed the form of large whorls of smooth membranes. In the distal segment the number of microvilli, vesicles, and vacuoles was less but mitochondria were more numerous than in the proximal part. Cells in the middle portion of the vas deferens showed some features of both proximal and distal parts. These structural characteristics, along with previous experiments, suggest that in the proximal portion of the vas deferens absorption of material from the lumen is an important function, while the cells of the distal part may synthesize steroids. Thus, these regional differences in ultrastructure probably reflect regional differences in function along the length of the vas deferens. After vasectomy the regional specializations were retained and did not appear to be altered. The possibility that this structural and functional sequence is significant for normal fertility and its relation to attempts to reconnect the vas deferens after vasectomy are considered.     

Opioid peptides and noradrenaline co-exist in large dense-cored vesicles from sympathetic nerve

The possibility that opioid peptides and noradrenaline co-exist not only in the desheathed bundle of bovine splenic nerve which contains ~98% sympathetic C-fibers, but also in the population of large dense-cored noradrenergic vesicles from these fibers, has been investigated. The primary fraction of large dense-cored vesicles which can be prepared at about 85% purity has been further subjected to density gradient and fractional centrifugation procedures, including D₂O-loading and unloading on modified second gradients, in an attempt to separate any minor population of particles which potentially could contain opioid peptides and contaminate the large dense-cored vesicle fraction. Measurement of opioid peptides, noradrenaline, dopamine and dopamine β-hydroxylase activity supports the conclusion that opioid peptides are stored in the primary population of large dense-cored vesicles per se, rather than in a minor population of contaminating particles from cells other than sympathetic C-fibers.This conclusion has implications for exocytotic release and the physiological role of the opioid peptides intra- and extra-neuronally. Nerve vesicle opioid peptides have a size less than 5000 daltons, in contrast to the high proportion of large peptides containing enkephalin sequences in the bovine adrenal medulla.     

Adenosine triphosphate mobilizes cytosolic calcium and modulates ionic currents in mouse taste receptor cells

By using photometry and the patch clamp technique, we identified P_2Y-like receptors in mouse taste receptor cells (TRCs) and found them to be coupled to Ca²⁺ mobilization and ionic current modulation. Particularly, adenosine triphosphate (ATP) and the P_2Y agonist 2-methylthio-ATP increased intracellular Ca²⁺ by stimulating the phosphoinositide pathway, whereas β,γ-methylene- -ATP, a P_2X agonist, was ineffective. In a distinctive TRC subpopulation, ATP closed Ca²⁺ channels. This regulation may underlie the negative feedback tuning neurotransmitter release. By mobilizing intracellular Ca²⁺, ATP activated Ca²⁺-dependent Cl⁻ channels, the intracellular event that may universally occur upon taste stimulation triggering IP₃ formation and Ca²⁺ release in the TRC cytoplasm.