Barbara R. Talamo

The association of octopamine with specific neurones along lobster nerve trunks.

Octapamine and its synthetic enzyme, tyramine beta‐hydroxylase (TBH), are found in high concentrations at two points along second thoracic nerve roots in lobsters. The first is in the proximal section of the second root between the ventral nerve cord and the bifurcation of the root into medial (to flexor muscles) and lateral (to extensors) branches. The second region of high concentration is within a well known crustacean neurosecretory system, the pericardial organ, located close to the ends of the lateral branches of the roots. 2. With several different staining procedures, small clusters of nerve cell bodies are found within the connective tissue sheath in the proximal regions of the second roots. No cell bodies are seen in the pericardial organ regions. Cell bodies are variable in number and position between corresponding roots in the same animal and homologous roots among different animals. The average numbers of cell bodies, however, correlate well with TBH and octopamine content, and with the synthesis of octopamine in these same regions of roots. 3. Small clusters of root cell bodies dissected from preparations have greater than 500‐fold higher activities of TBH than isolated efferent excitatory and inhibitory or afferent sensory axons. 4. Along with octopamine, the preferential synthesis of acetylcholine and serotonin is also seen in proximal segments of roots. Acetylcholine synthesis in these regions may represent transmitter synthesized in the nerve terminals innervating the root cells. The role of serotonin in these regions is not understood at this time but the amounts of endogenous serotonin found are only a tenth of the amounts of octopamine present. 5. Dopamine is not synthesized from tyrosine in second thoracic roots. However, if DOPA or dopamine are used as precursor compounds, then noradrenaline, which is usually not found in lobsters, can be accumulated in proximal segments of roots. 6. Phenolamines are converted to two further metabolites by lobster tissues. The compounds are unidentified and are named fast and slow product on the basis of their migration on electrophoresis at acid pH. Some partial characterization of slow product reveals that it is a mixture of compounds that can be converted on mild acid hydrolysis to fast product and the parent phenolamine. 7. The several lines of evidence presented suggest that nerve cells found in the proximal segments of the second thoracic roots contain and can synthesize octopamine. Since not all the cells in any single root have been analysed for octopamine or TBH, however, the possibility that one or more of the cells contain physiologically interesting substances other than octopamine is not eliminated.

Octopamine release at two points along lobster nerve trunks.

Nerve cells in the proximal regions of second thoracic roots in lobsters have been injected with the fluorescent dye Procion Yellow. Examination of the roots reveals an elaborate array of cell branches in a superficial layer of the root in the vicinity of the cell bodies. Large varicosities, up to 10 mum in diameter, are seen lined up along fine nerve branches. 2. In these same regions, electron microscopic examination shows the presence of large profiles filled with 0‐1‐0‐2 mum dense cored granules, and having the appearance of nerve endings. These profiles probably correspond to the varicosities seen in the Procion Yellow injections. The dense cored granules within the endings have a crystalline substructure. All the endings are found within 7 mum of the surface of the root and no obvious physiological target tissue exists in their surroundings. Endings have not been traced directly to root cell bodies.However, granules of similar dimensions to those seen in endings are found in cell bodies, axon‐hillock regions and numerous axonal profiles in the superficial root regions near cell bodies. The morphological studies suggest that the root neurones have the typical appearance of neurosecretory cells. 3. Octopamine pools in cell body regions of second thoracic roots can be isotopically labelled by incubation with either [3H]tyramine or [3H]‐tyrosine. After labelling, pulsing with 100 mM potassium causes an increase in the rate of release of radioactive material. Upon return to normal media background rates of release are re‐established. The enhanced efflux has the following properties: (a) repeated pulses of potassium release less radio‐active material each time; (b) a prolonged potassium pulse produces first a peak of release, then a decline to a plateau, and the plateau level of release is maintained for the duration of the potassium pulse; (c) release is dependent on the presence of calcium ions in the bathing fluid and 40 mM cobalt prevents release; (d) release is selective for octopamine. With tyrosine as a precursor compound, as much radioactive tyrosine as octopamine is found in tissues after incubation, yet pulsing with potassium causes an enhanced efflux only of octopamine from preparations. 4. Release of octopamine also can be demonstrated from pericardial organs near the ends of lateral branches of the roots and the properties of the release are identical to those seen with cell body regions. 5. Physiological studies, in which root cells are antidromically activated while recording from cell bodies, suggest that the distal endings of at least some of the root cells are at the pericardial organs. 6. The results suggest that root cell neurones are neurosecretory cells capable of releasing octopamine at two points: one near cell bodies, the other at the pericardial organs near the distal ends of the roots...

Octopamine neurons: morphology, release of octopamine and possible physiological role

Octopamine is a major amine derived from tyrosine in the lobster nervous system ~,15. It is also found in the nervous systems of various other invertebrates 6,7:1° and mammals 8 but its function is unknown. In Aplysia, single neurons with a high endogenous content of octopamine have been identified li, and octopamine produces a hyperpolarization when iontophoretically applied to certain neurons in central ganglia 5. In a previous communication we reported the unique association of octopamine with groups of neurons along the proximal portions of thin second roots leaving the lobster thoracic ganglia 1-5. The cells are generally located close to the point where these roots divide into a lateral branch innervating extensor muscles and a medial branch innervating flexor muscles. The muscles are continuations into the thorax of the main abdominal musculature involved in the tail flip. In this communication we report the fine structural anatomy of processes of the octopamine cells in regions near their cell bodies, the release of octopamine from these regions with K ~ depolarization and the induction by octopamine of a long lasting contracture in lobster exoskeletal muscles. Our previous studies with neutral red and osmium staining of cells indicated that cell processes might be located in the same regions of roots where the cell bodies are found. Accordingly, cells were injected with the dye Procion Yellow to reveal further details o f their geometry. In Fig. 1, a whole mount of a first thoracic root with two injected cells is shown. Fine processes of the two cells branch extensively in the vicinity of the cells. Focussing up and down in the fluorescence microscope reveals that most branches are in a superficial layer of the root. Deeper in the root, where axons of other neurons run, fewer branches are seen. The packing of processes in Fig. 1 is sufficiently dense that if all 12 cells in the first root had been injected, and these cells also had similar wide ramifications of branches, one would expect the entire superficial layer to be packed with cellular processes. Therefore, electron microscopic examination of such regions should readily detect processes of the root cells.

Salivary Gland P2 Nucleotide Receptors

The effects of ATP on salivary glands have been recognized since 1982. Functional and pharmacological studies of the P2 nucleotide receptors that mediate the effects of ATP and other extracellular nucleotides have been supported by the cloning of receptor cDNAs, by the expression of the receptor proteins, and by the identification in salivary gland cells of multiple P2 receptor subtypes. Currently, there is evidence obtained from pharmacological and molecular biology approaches for the expression in salivary gland of two P2X ligand-gated ion channels, P2Z/P2X7 and P2X4, and two P2Y G protein-coupled receptors, P2Y1 and P2Y2. Activation of each of these receptor subtypes increases intracellular Ca2+, a second messenger with a key role in the regulation of salivary gland secretion. Through Ca2+ regulation and other mechanisms, P2 receptors appear to regulate salivary cell volume, ion and protein secretion, and increased permeability to small molecules that may be involved in cytotoxicity. Some localization of the various salivary P2 receptor subtypes to specific cells and membrane subdomains has been reported, along with evidence for the co-expression of multiple P2 receptor subtypes within specific salivary acinar or duct cells. However, additional studies in vivo and with intact organ preparations are required to define clearly the roles the various P2 receptor subtypes play in salivary gland physiology and pathology. Opportunities for eventual utilization of these receptors as pharmacotherapeutic targets in diseases involving salivary gland dysfunction appear promising.

Expression and Trans-synaptic Regulation of P2x4 and P2z Receptors for Extracellular ATP in Parotid Acinar Cells EFFECTS OF PARASYMPATHETIC DENERVATION

Trans-synaptic regulation of muscarinic, peptidergic, and purinergic responses after denervation has been reported previously in rat parotid acinar cells (McMillian, M. K., Soltoff, S. P., Cantley, L. C., Rudel, R., and Talamo, B. R. (1993) Br. J. Pharmacol. 108, 453–461). Characteristics of the ATP-mediated responses and the effects of parasympathetic denervation were further analyzed through assay of Ca2+ influx, using fluorescence ratio imaging methods, and by analysis of P2x receptor expression. ATP activates both a high affinity and a low affinity response with properties corresponding to the recently described P2x4 and the P2z (P2x7)-type purinoceptors, respectively. Reverse transcription-polymerase chain reaction analysis reveals mRNA for P2x4 as well as P2x7 subtypes but not P2x1, P2x2, P2x3, P2x5, or P2x6. P2x4 protein also is detected by Western blotting. Distribution of the two types of ATP receptor responses on individual cells was stochastic, with both high and low affinity responses on some cells, and only a single type of response on others. Sensitivity to P2x4-type activation also varied even among cells responsive to low concentrations of ATP. Parasympathetic denervation greatly enhanced responses, tripling the proportion of acinar cells with a P2x4-type response and increasing the fraction of highly sensitive cells by 7-fold. Moreover, P2x4 mRNA is significantly increased following parasympathetic denervation. These data indicate that sensitivity to ATP is modulated by neurotransmission at parasympathetic synapses, at least in part through increased expression of P2x4mRNA, and suggest that similar regulation may occur at other sites in the nervous system where P2x4 receptors are widely expressed.

Two distinct cytosolic calcium responses to extracellular ATP in rat parotid acinar cells.

1 Increasing concentrations of ATP (0.5 μm‐300 μm) produced a biphasic increase in intracellular calcium concentration [Ca]i in rat parotid acinar cells, reflecting two distinct Cai responses to extracellular ATP. 2 In the absence of Mg2+ (with 3 mm CaCl2 in the buffer solution), the more sensitive response was maximal at 3–5 μm and was not further increased by 30 μm ATP. This response to ATP was not well maintained and was blocked by ADP (0.5 mm). A second, much larger increase in Cai was observed on addition of 300 μm ATP. This larger effect, which we have described previously, appears to be mediated by ATP4–, and was selectively reversed by 4,4′‐di‐isothiocyanato‐dihydrostilbene‐2,2′‐disulphonate as well as by high concentrations of α,β‐methylene ATP. 3 Among ATP analogues, only the putative P2Z agonist, 3′‐0‐(4‐benzoyl)benzoyl‐ATP distinguished between the two responses. This analogue was at least 10 fold more potent than ATP in stimulating the ATP4–‐response, but did not evoke the more sensitive response. The agonist potency series for both responses to ATP was identical for other analogues examined (ATP>ATP‐γS = 2‐methylthio ATP (a P2y‐selective agonist) 72>ADP, ITP and α,β‐methylene ATP (a P2X‐selective agonist)). 4 Although the effect of ATP4– could best be characterized as a P2Z‐type purinoceptor response, this effect was strongly and selectively blocked by reactive blue 2, a putative P2y‐purinoceptor antagonist. Reactive blue 2 may bind to and block P2Z purinoceptors since [γ32P]‐ATP binding to parotid cells was inhibited by this compound. 5 In contrast to the response to ATP4–, the more sensitive response to ATP was potentiated by reactive blue 2 and was less affected by increases in external Mg2+ and Ca2+. 6 Parasympathetic denervation selectively increased the more sensitive response, suggesting that it may be physiologically regulated.

Coomassie Brilliant Blue G is a more potent antagonist of P2 purinergic responses than Reactive Blue 2 (Cibacron Blue 3GA) in rat parotid acinar cells

The ability of Brilliant Blue G (Coomassie Brilliant Blue G) and Reactive Blue 2 (Cibacron Blue 3GA) to block the effects of extracellular ATP on rat parotid acinar cells was examined by evaluating their effects on ATP-stimulated 45Ca2+ entry and the elevation of [Ca2+]i (Fura 2 fluorescence). ATP (300 microM) increased the rate of Ca2+ entry to more than 25-times the basal rate and elevated [Ca2+]i to levels more than three times the basal value. Brilliant Blue G and Reactive Blue 2 greatly reduced the entry of 45Ca2+ into parotid cells, but the potency of Brilliant Blue G (IC50 approximately 0.4 microM) was about 100-times that of Reactive Blue 2. Fura 2 studies demonstrated that inhibitory concentrations of these compounds did not block the cholinergic response of these cells, thus demonstrating the selectivity of the dye compounds for purinergic receptors. Unlike Reactive Blue 2, effective concentrations of Brilliant Blue G did not substantially quench Fura 2 fluorescence. The greater potency of Brilliant Blue G suggests that it may be very useful in identifying P2-type purinergic receptors, especially in studies which utilize fluorescent probes.

Elevation of [Ca2+]i and the Activation of Ion Channels and Fluxes by Extracellular ATP and Phospholipase C-Linked Agonists in Rat Parotid Acinar Cells

Extracellular ATP initiates a variety of changes in the parotid acinar cell. The initial effect appears to be the entry of Ca2+ (and perhaps Na+), and a series of ion transport events result from the subsequent elevation of [Ca2+]i. Agonists of phospholipase C-linked receptors elevate [Ca2+]i by a different pathway, involving the generation of inositol polyphosphate compounds, but share in the subsequent initiation of the ion transport events. Although the maintenance of the physiological changes may depend on specific inositol polyphosphate intermediates, the critical initiating factor is the elevation of [Ca2+]i. Fluid secretion by the parotid gland is triggered by the action of neurotransmitters, which alter the membrane permeability of the acinar cell. The similarities between the two receptor-mediated activation pathways suggests that ATP may act as a neurotransmitter and play a role in the control of fluid secretion. Basing our analysis on the purinoceptor characteristics outlined by Gordon, we suggest that the parotid receptor belongs to the P2Z class, which is highly sensitive to ATP4-. Basing his analysis on the earlier report by Gallacher of the effects of ATP on mouse parotid cells, Gordon placed the parotid purinoceptor in a different P2 subclass (P2Y). However, our findings of an increased potency of ATP in the absence of Mg2+, as well as the potency order of different nucleotides, indicate that the P2Z class is a more appropriate category.

A new assay for fatty acid desaturation.

Abstract The enzymic formation of unsaturated fatty acids from their saturated homologs is ordinarily assayed by gas-liquid chromatography or by chromatography of the complexes formed between the olefinic acids and mercuric acetate or silver nitrate. In the present paper a rapid, sensitive assay is described. It is based on the release of tritium as tritiated water from a substrate specifically labeled at the positions to be desaturated. The method circumvents the time consuming operations (saponification, extraction, esterification, and chromatography) of the older procedures. A similar principle has been utilized for assaying aromatic hydroxylation (1). Both CoA and acyl carrier protein (ACP) fatty acid thioesters are precipitable by acid and can be collected on a Millipore filter which is conveniently attached to a syringe. The tritiated water filtrate is collected and an aliquot counted. The sensitivity of the method extends to the picomole range and is limited only by the specific activity of the substrate.

Rapid desensitization of substance P- but not carbachol-induced increases in inositol trisphosphate and intracellular Ca++ in rat parotid acinar cells

The effects of supramaximal concentrations of substance P and the cholinergic agonist carbachol on the accumulation of inositol trisphosphate and the elevation of the intracellular free calcium concentration were compared in rat parotid acinar cells. Substance P was fully as effective as carbachol at initial times, but there was a rapid loss of the substance P responses while the effects of carbachol were well maintained. The loss of the substance P responses represented desensitization rather than degradation of the peptide since further additions of substance P were without effect. Desensitization to substance P did not involve long-term loss of substance P receptors as it was fully reversible in less than twenty minutes, the minimum time to extensively wash previously desensitized cells.