Innervation of the American cockroach salivary gland: neurophysiological and pharmacological investigations

Application of 5-hydroxytryptamine to the gland in vitro in concentrations as low as 10^?12 M effected continuous secretion of fluid. This suggests that 5-HT or a related compound may be the neurotransmitter substance. In vivo injections of p-chorophenylalanine did not affect secretion. Applications of pilocarpine and acetylcholine had only a transitory effect upon secretions. Nerve-section studies implicate the salivary duct nerves coming from the suboesophageal ganglion as motor nerves controlling secretion. The oesophageal salivary nerves from the brain were not severed due to excessive trauma. Simultaneous electrical recordings of the salivary nerves show no common activity. Nerve section demonstrates that the activity in these nerves is efferent. Central inhibition of nervous activity occurs during sensory stimulation, etc. Electrical stimulation of the salivary duct nerve in vitro effects salivary secretion for several hours; however, the loss of secretion is not due to failure of the nerves, but to unknown factors. Histological sections of the stimulated glands failed to show cytological changes.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Oxidative Stress and Ca2+ Signals Involved on Cadmium-Induced Apoptosis in Rat Hepatocyte

Cadmium (Cd) is an important industrial and environmental pollutant. In animals, the liver is the major target organ of Cd toxicity. In this study, rat hepatocytes were treated with 2.5～10 μM Cd for various durations. Studies on nuclear morphology, chromatin condensation, and apoptotic cells demonstrate that Cd concentrations ranging within 2.5～10 μM induced apoptosis. The early-stage marker of apoptosis, i.e., decreased mitochondrial membrane potential, was observed as early as 1.5 h at 5 μM Cd. Significant (P?P?2+ concentration ([Ca²⁺] _i ) of Cd-exposed cells significantly increased (P?2+] _i may play an important role in apoptosis. Overall, these results showed that oxidative stress and Ca²⁺ signaling were critical mediators of the Cd-induced apoptosis of rat hepatocytes.     

Low-level laser therapy with 850 nm recovers salivary function via membrane redistribution of aquaporin 5 by reducing intracellular Ca2+ overload and ER stress during hyperglycemia

The overall goal is to study the effect of low-level laser therapy (LLLT) on membrane distribution of major water channel protein aquaporin 5 (AQP5) in salivary gland during hyperglycemia. Par C10 cells treated with high glucose (50?mM) showed a reduced membrane distribution of AQP5. The functional expression of AQP5 was downregulated due to intracellular Ca²⁺ overload and ER stress. This reduction in AQP5 expression impairs water permeability and therefore results in hypo-salivation. A reduced salivary flow was also observed in streptozotocin (STZ)-induced diabetic mice model and the expression of AQP5 and phospho-AQP5 was downregulated. Low-level laser treatment with 850?nm (30?mW, 10?min?=?18?J/cm²) reduced ER stress and recovered AQP5 membrane distribution via serine phosphorylation in the cells. In the STZ-induced diabetic mouse, LLLT with 850?nm (60?J/cm²) increased salivary flow and upregulated of AQP5 and p-AQP5. ER stress was also reduced via downregulation of caspase 12 and CHOP. In silico analysis confirmed that the serine 156 is one of the most favorable phosphorylation sites of AQP5 and may contribute to the stability of the protein. Therefore, this study suggests high glucose inhibits phosphorylation-dependent AQP5 membrane distribution. High glucose induces intracellular Ca²⁺ overload and ER stress that disrupt AQP5 functional expression. Low-level laser therapy with 850?nm improves salivary function by increasing AQP5 membrane distribution in hyperglycemia-induced hyposalivation.     

The dependence of fluid secretion by mandibular salivary gland and pancreas on extracellular calcium

Acetylcholine-stimulated fluid secretion from the perfused rabbit mandibular salivary gland was inhibited in a biphasic manner when extracellular calcium concentration was reduced in the range 5 X 10(-4) - 10(-5)M. An initial rapid inhibition was followed by partial recovery to a plateau, the level of which depended upon the calcium concentration. Since no recovery was observed during substitution of calcium by strontium, recovery may depend upon an increased membrane permeability to calcium. It is concluded that acetylcholine evokes fluid secretion in this gland by enhancing calcium entry from the extracellular space, an action which can be mimicked by the calcium ionophore A23187. Changes in the electrolyte composition of saliva during calcium-depletion were such as to suggest that ductal reabsorption of sodium and chloride, and secretion of potassium are inhibited as extracellular calcium concentration is reduced. Secretin-stimulated fluid secretion from the cat pancreas was unaffected when perfusate calcium concentration was reduced to 2.5 X 10(-6)M and carbachol-stimulated amylase secretion was only slightly reduced. Since the latter is a calcium-dependent process, the source of calcium is presumably intracellular. In both glands, reducing calcium to 1 X 10(-6)M caused rapid and irreversible inhibition of fluid secretion.     

Control by calcium of protein discharge and membrane permeability to potassium in the rat lacrimal gland

Discharge of protein from slices of rat exorbital lacrimal gland was stimulated by 10^?5 M carbachol. This response was blocked by 10^?4 M atropine or by the omission of extracellular calcium. In the latter case, secretion could be restored by the reintroduction of calcium to the medium. Carbachol (10^?5 M) also stimulated the release of ⁸⁶Rb (a marker for potassium) from the slices. This effect was completely blocked by 10^?4 M atropine. The initial transient release of ⁸⁶Rb was only partially inhibited by Ca removal, but the later sustained phase of release was completely blocked. As with protein secretion, this effect of Ca removal could be reversed by re-introduction of Ca to the medium. It is concluded that activation of cholinergic receptors in the lacrimal gland stimulates protein discharge and increases potassium permeability by mechanisms utilizing extracellular calcium ions.     

Calcium fluxes accompanying the action of 5-hydroxytryptamine on mussel hearts

1. Isolated, superfused ventricles of Geukensia demissa demissa were soaked in artificial seawater containing radiocalcium (⁴⁵Ca). The efflux of ⁴⁵Ca into cold seawater was analyzed, and the effect of 5-hydroxytryptamine (5-HT) on the efflux was studied.2. The control ⁴⁵Ca efflux contained three identifiable components with the following time constants: τ₁ = 2.27 ± 0.30 min; τ₂ = 8.8 ± 1.1 min; and τ₃ = 133.1 ± 39.2 min.3. Excitation by 5-HT (10⁻⁶ M) caused an immediate systolic contracture and a transient increase in ⁴⁵Ca efflux. The source of this efflux was depleted by a single, 10 min episode of 5-HT excitation.4. When 5-HT was washed out, an efflux increase of long duration was observed. The time constant (9.3 ± 1.8 min) was identical to that of the second control component, τ₂.5. 5-HT caused excitation by decreasing the net calcium efflux, thereby increasing intracellular free calcium. No such change was associated with cardioinhibition by 5-HT.     

Stimulation of 45Ca efflux from rat lacrimal gland slices by carbachol and epinephrine

The effects of carbachol (10^?5M) and epinephrine (10^?5M) on efflux of ⁴⁵Ca from rat exorbital lacrimal gland slices were examined. Both carbachol and epinephrine stimulated a transient release of ⁴⁵Ca from the tissue. The quantity of Ca released was estimated to be of the order of 0.5 μmol/g. Release of ⁴⁵Ca by one agonist prevented subsequent release of ⁴⁵Ca by a different agonist. These data support the hypothesis put forth previously that in the lacrimal gland muscarinic or α-adrenergic receptor activation causes a transient increase in membrane permeability to K by triggering the release of a sizable intracellular pool of Ca common to both receptors.     

Tryptamine as an endogenous modulator of neuronal sensitivity to serotonin

Research was performed on sensory ganglia isolated from adult rats using intracellular techniques for recording membrane potential and by measuring resistance at the membrane of individual units. It was found that tryptamine at high concentrations manifests serotoninlike activity, but, at concentrations not affecting potential and resistance at the neuronal membrane, either reinforces (at a concentration of 10^–7 M) or attenuates (at 10^–5 M) serotonin (5-HT) effects mediated by type 1A (but not type 2) 5-HT receptors. 5-HT-modulated effects were produced by tryptamine-induced changes in 5-HT sensitivity at the neuronal membrane and remained unchanged by maximum level of this transmitter. Harmane acts similarly to tryptamine, although harmane derivatives (C-412 and C-506 respectively) produce either potentiation or inhibition of 5-HT_1A over the entire concentration range used (of 10^–7 M-10^–5 M). The allosteric nature of 5-HT-modulation by tryptamine and harmane is discussed.M. Gor'kii Medical Institute, Donetsk, Ukrainian SSR. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 352–357, May–June, 1989.     

POSTSYNAPTIC ADRENERGIC-CYCLIC AMP CONTROL OF THE SEROTONIN CONTENT OF CULTURED RAT PINEAL GLANDS

Abstract— This investigation was designed to determine whether the amount of serotonin (5-HT) in cultured pineal glands can be altered by norepinephrine (NE). Treatment with l -NE (10^?5-10^?7m ) for 4-6 h caused a gradual decrease in the concentration of 5-HT to a value that was less than 30% of that in the untreated control gland. This effect was observed using chronically denervated pineal glands. d -Norepinephrine (10^?6-10^?7m ) and dopamine (10^?4m ) were ineffective in lowering 5-HT. The effect of l -NE was completely blocked by a β-adrenergic receptor blocker, propranolol and was only slightly decreased by α-adrenergic receptor blockers. These observations indicate that l -NE acts post-synaptically via a highly specific β-adrenergic mechanism. The effect of l -norepinephrine was mimicked by theophylline and N⁶, 2′0-dibutyryl adenosine 3′,5′-monophosphate, an indication that adenosine 3′,5′-monophosphate is involved in the effect of l -NE on 5-HT. Treatment with cycloheximide, which by itself caused a decrease in pineal 5-HT, also blocked any further decrease caused by treatment with l -NE, an indication that protein synthesis is necessary for maintenance of baseline levels of serotonin and for the effect of l -NE to be observed. The total amount of l -[³H]NE and degradation products of L-[³H]NE in the gland after 6 h of treatment with l -[³H]NE was less than 3 pmol. This amount of l -NE and degradation products of l -NE could not account for the decrease of 100-200 pmol of 5-HT on the basis of a mole for mole replacement of 5-HT by l -NE. These findings are consistent with the hypothesis that non-neuronal pineal 5-HT is physiologically regulated by the release of l -NE from the sympathetic nerve network.     

Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle

This report demonstrates that the commonly used anesthetic agent, pentobarbital sodium, in concentrations of 1 · 10^?4 to 2 · 10^?3 M inhibits calcium (Ca²⁺) uptake in both rat aortic and portal venous smooth muscle. The data indicate that total exchangeable Ca²⁺ in portal vein is reduced by about 15% in 1 · 10^?4 M pentobarbital sodium, while the intracellular exchangeable Ca²⁺ is reduced by 24%. On the other hand, in aortic smooth muscle, while 5–20 · 10^?4 M pentobarbital sodium reduces total exchangeable Ca²⁺ by about 15%, intracellular Ca²⁺ is reduced by 22% in 5 · 10^?4 M pentobarbital sodium and by 38% in 2 · 10^?3 M pentobarbital sodium. The present studies thus reveal that concentrations of pentobarbital sodium known to be present during induction of surgical anesthesia can exert significant inhibitory effects on exchangeability and transmembrane movement of Ca²⁺ in at least two different types of blood vessels.     

Extracellular calcium dependence of contracture and modulation by serotonin in buccal muscle E1 of Aplysia

Serotonin [5-hydroxytryptamine (5-HT)] enhances acetyl choline (ACh)-elicited contractures of Aplysia buccal muscles E1 and I5. The possible role of external calcium in regulating the magnitude of ACh contracture in the presence and absence of 5-HT was investigated. Superfusion of E1 with zero calcium medium caused ACh contractures to fail within one to two minutes. Recovery of ACh contracture upon restoring normal medium occurred within two to four minutes. In the absence of 5-HT, ACh contracture decreased proportionally to external [Ca⁺⁺] in the concentration range of 0–10 mM; however, the amount of enhancement of of ACh contracture following 5-HT treatment did not decrease with external [Ca⁺⁺] as long as [Ca⁺⁺] was above a threshold concentration that varied from preparation to preparation. For most preparations, the enhancement of ACh contracture by 5-HT was dependent on the presence of external calcium during 5-HT treatment. Calcium influx into muscles E1 and I5 increased approximately two and a half fold in the presence of 10^?6 M 5-HT. A model in which 5-HT brings about calcium “loading” of an ACh releasable intracellular storage site is discussed.     

Inhibition of the positive inotropic effect of anthopleurin-A (AP-A) by dantrolene

The effect of dantrolene on the positive inotropic effects (PIE) of three cardiotonic agents was assessed on rat and rabbit atria. Dantrolene (10^?5M) had no effect on contractile tension or on the PIE to isoproterenol (10^?10?10^?7M) or ouabain (10^?6?10^?4). The dose-response curve for the PIE of anthopleurin-A (AP-A) was shifted to the right in rat and rabbit atria by dantrolene (10^?4?10^?6M). The maximum effect and the concentration of AP-A required for it remained the same. The results suggest that the PIE of AP-A involves intracellular translocation of calcium, unlike those of isoproterenol and ouabain which require increased transmembrane calcium flux. This conclusion is supported by the observation that the exchange and distribution of the labile calcium involved in excitation-contraction coupling was unaffected by AP-A (10^?8M), by dantrolene (10^?6M) or by the combination. Therefore, AP-A may produce its cardiotonic effect by a action at an intracellular site, a mechanism unlike that of isoproterenol or ouabain.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

The inhibition of the serum-stimulated increase of ornithine decarboxylase by ionophores and local anesthetics

The addition of fresh serum-containing growth medium to L1210 mouse leukemic cells in culture resulted in a 5-fold increase in ornithine decarboxylase (l-ornithine carboxy-lyase, EC 4.1.1.17) activity. The presence of microtubule disrupting agents (colchine, vinblastine) or cations (5–10 mM K⁺, Na⁺ or Mg²⁺) abolishes this increase of ornithine decarboxylase activity (Chen, K.Y., Heller, J.S. and Canellakis, E.S. (1976) Biochem. Biophys. Res. Commun. 70, 212–219). Based on these observations we proposed that fluctuation in cellular cation concentrations may act as a link between the membrane structure and ornithine decarboxylase. To test this proposal, we studied the effects of selective membrane perturbing agents such as ionophores and local anesthetics, on the serum-stimulated increase of ornithine decarboxylase activity in L1210 cells. Among the six inonophores tested, valinomycin was the most potent one, with I₅₀ value (concentration that gives 50% inhibition of orthinine decarbocylase activity) of 6·10^?9 M. Dibucaine and tetracaine were also effective inhibitors at 10^?4?10^?5 M. The I₅₀ values of valinomycin on the protein synthesis and RNA synthesis, however, were greater than 1·10^?6 M. These results substantiate the notion that ornithine decarboxylase activity can be regulated at plasma membrane level and such regulation is related to the perturbation of cellular cation pools.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

Electrical responses of salivary gland cells of the gastropod mollusc Philine aperta to putative neurotransmitters—II. Octopamine, 5-HT and other biogenic amines

1. Intracellular recordings were made from salivary gland cells of the mollusc Philine aperta. Salivary cell action potentials were produced in a dose-dependent manner in response to bath-application of octopamine, 5-hydroxytryptamine (5-HT) and certain related biogenic amines.2. Evidence that amines act on presynaptic receptors on nerve terminals rather than directly on gland cell receptors is presented. These receptors may serve to modulate salivary neuroglandular transmission.3. Octopamine and 5-HT action was reversibly blocked or reduced by the antagonists phentolamine, chlorpromazine, cyproheptidene, yohimbine, D-tubocurarine, hexamethonium and atropine.     

Adult development of the hippocampal-serotonin system of C57BL/6N mice; analysis of high-affinity uptake of 3H-5HT in slices and synaptosomes

The saturable and specific high-affinity uptake of [³H]serotonin ([³H]5HT) (5 × 10^?8 M) was studied in slices from the hippocampus, parietal cortex, septum-preoptic area, and hypothalamus of male 2, 6, 12 and 24–32 month old C57BL/6N mice. Hippocampal [³H]5-HT uptake showed a significant biphasic relationship to age, with lower values in the 2 and 24–32 month old mice compared to 6 month old mice. No significant age effects were seen in the other regions, or in [³H]norepinephrine high-affinity uptake in the hippocampus.Studies of the high-affinity uptake mechanism in synaptosomal preparations were made in a subgroup of 12 and 24 month old mice. A micro-assay using a tissue-harvester measured high-affinity uptake on 8–30 μl of the P₂ suspension (crude-synaptosomal preparation). The high-affinity uptake was linear for 4 min at 37°C and inhibited in both the adult and aged tissue by 10^?5 M cold 5-HT (83 and 78% respectively), 10^?5 M fluoxetine (85 and 82% respectively) and 10^?3 M NaCN (57 and 57% respectively). Kinetic analysis of the [³H]5HT high-affinity uptake in the hippocampus (3 min, 37°C) revealed the same apparent K_m for serotonin at both ages (6.7 x 10^?8 M), but a 44% decrease in V_max in the aged hippocampal synaptosomal high-affinity uptake compared to adults (120 vs 215 pmol of 5-HT/g-tissue/3 min).These results are discussed in relationship to the reported age effects on the intrinsic neurons of the hippocampus.     

Antibody-dependent lymphocyte mediated cytotoxicity mechanism and modulation by cyclic nucleotides.

The injury to antibody coated thymocytes by the “K cell” among nonsensitized rat splenocytes was modulated by altering the cellular level of cAMP and cGMP. Preincubation of the attacking cell population with 1 μg/ml cholera toxin caused an elevation of cAMP levels of 1–18 pM per 10⁷ cells and was associated with a proportionate reduction in cytotoxicity. Agents which are known to raise cAMP levels including the Prostaglandins (PG) E₁ (10^?7–10^?5 M), PGF_2α (10^?5–10^?3 M), PGA₁ (10^?9–10^?5M), and theophylline (10^?3 M) also produced marked suppression of antibody dependent lymphocyte mediated cytotoxicity. Direct elevation of cellular levels of cGMP by the analog 8-bromo cGMP (5 × 10^?6M) led to augmentation of cytotoxicity. Removal by EDTA and MgEDTA of calcium and magnesium ions from the culture media markedly inhibited cytotoxicity.     

Investigation of the effect of intracellular calcium on the cAMP-mediated increase in the calcium current

The experiments were conducted on intracellularly persfused neurons of the molluskHelix pomatia, in which a serotonin (5-HT)-induced increase in the calcium current (I_Ca), mediated by cAMP, is observed. It was established that desensitization of the cell to the action of 5-HT is due to an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). At [Ca²⁺]_i=10^–7 and 10^–6 M, the effect of 5-HT constituted 60 and 32% of this value in the control (in the presence of 10 mM EGTA in the intracellular solution), respectively; at [Ca²⁺]_i=10^–5 M, there was no effect of 5-HT at all. The addition of the calmodulin antagonist trifluoperazine (5·10^–5 mM) or blockers of phosphodiesterase (5 mM theophylline or 10^–4 M IBMX) to the perfusate sharply weakened the ability of calcium to inhibit the effect of 5-HT; at [Ca²⁺]_i=10^–5 M, in the presence of one of these compounds, the effect constituted 60–70% of its control value. It is concluded that the calcium-calmodulin-dependent phosphodiesterase is a key link in the interaction of the two-signal transduction pathway — Ca²⁺ and cAMP in modulation of the calcium-channel activity.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 306–313, May–June, 1991.