cyclic-gmp and 4-5-diaminofluorescein

Insects, like other animals, require sodium chloride (NaCl) as part of their normal diet and detect it with contact chemoreceptors on the body surface. By adjusting the responsiveness of the chemosensory neurons within these receptors insects can modify the intake of salt and other nutrients, and it has been hypothesized that the responsiveness of chemosensory neurons is regulated by nitric oxide (NO). To identify potential sources of NO in the periphery, the authors applied the NO-sensitive fluorescent probe 4,5-diaminofluorescein and the universal NO synthase antibody, and found that in locusts NO is synthesized within one particular class of cells of the epidermis, the glandular cells, from where it may diffuse to neighboring chemosensory neurons. The effects of NO on chemosensory neurons were investigated by recording from contact chemoreceptors on the leg while perfusing it with drugs that interfere with NO signaling. Results showed that both endogenous and exogenous NO decreased the frequency of action potentials in chemosensory neurons in response to stimulation with NaCl by acting via a cyclic guanosine monophosphate-independent pathway. Variation of the NaCl concentration in the perfusion solution demonstrated that the synthesis of NO in glandular cells depends on the NaCl concentration in the hemolymph. By contrast NO increased the frequency of action potentials in chemosensory neurons in response to sucrose stimulation. The authors suggest that NO released from glandular cells modulates the responsiveness of chemosensory neurons to regulate NaCl intake, and hypothesize that NO may play a key role in the signaling of salt and sugars.

Topics: Action Potentials; Analysis of Variance; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Epidermal Cells; Epidermis; Fluorescein; Grasshoppers; Hydrazines; Male; Neurons, Afferent; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Signal Transduction; Sodium; Sodium Chloride; Taste

The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production using the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2). We initiated investigations by adding NO from an external source by means of the NO-donor, S-nitroso-N-acetyl-penicillamine (SNAP). Cellular concentrations of cyclic guanosine 5'-phosphate (cGMP) ([cGMP]) were measured by radioimmunoassay (RIA), and we found that SNAP induced a fast increase in the [cGMP], amounting to 350% of the [cGMP] in resting cells. Moreover, addition of SNAP and elevating [cGMP] in fura-2 loaded lacrimal acinar cells, resulted in a cGMP-dependent protein kinase-mediated release of Ca2+ from intracellular stores, leading to a rise in the intracellular free Ca2+ concentration ([Ca2+]i). The Mn2+ quenching studies revealed that the Ca2+ release was not accompanied by Ca2+ influx. Finally, we demonstrate that lacrimal acinar cells possess endogenous NOS activity, which is activated by beta-adrenergic stimulation and not by a rise in [Ca2+]i alone. We show that in rat lacrimal acinar cells, NO and cGMP induce Ca2+ release from intracellular stores via G kinase activation. However, the changes in [Ca2+]i are relatively small, suggesting that this pathway plays a modulatory role in Ca2+ signalling, thus not by itself causing fast transient increases in [Ca2+]i. In addition, we suggest that endogenously produced NO activated by beta-adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.

Topics: Animals; Calcium; Cyclic GMP; Fluorescein; Fura-2; Indicators and Reagents; Lacrimal Apparatus; Male; Nitric Oxide; Nitric Oxide Synthase; Organ Culture Techniques; Penicillamine; Radioimmunoassay; Rats; Rats, Wistar; Signal Transduction; Time Factors

The activity of nitric oxide synthase (NOS) in rat parotid acinar cells was measured using a newly synthesized fluorescent NO indicator DAF-2/DA. Our results show that NO production is most effectively stimulated by activation of the beta-adrenergic receptor, and to a minor extent by substance P (SP). NO activates the production of cGMP, an intracellular messenger that has been shown to release Ca2+ from ryanodine-sensitive intracellular stores. We found that cGMP is also able to release Ca2+ from ryanodine-insensitive intracellular stores. Our data show that a rise in the cGMP concentration induces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] synthesis and Ca2+ release from intracellular stores.

Topics: Adrenergic beta-Agonists; Animals; Calcium; Cyclic GMP; Fluorescein; Indicators and Reagents; Inositol Phosphates; Isoproterenol; Male; Nitric Oxide; Parotid Gland; Rats; Rats, Wistar; Substance P

We investigated the cellular regulation of nitric oxide synthase (NOS) activity in isolated acinar cells from rat parotid and human labial salivary glands, using the newly developed fluorescent nitric oxide (NO) indicator, DAF-2. We found that sympathetic stimulation with norepinephrine (NE) caused a strong increase in NO synthesis that was not seen after parasympathetic stimulation with acetylcholine. In rat parotid acinar cells, we furthermore investigated to which extent the NOS activity was dependent on the intracellular free Ca2+ concentration ([Ca2+]i) by simultaneously measuring NO synthesis and [Ca2+]i. It was found that a simple correlation between the rise in [Ca2+]i and the rate of NO production following NE stimulation does not exist, and studies in which [Ca2+]i was elevated by means of the Ca2+ ionophore, ionomycin, further established that even a very large rise in [Ca2+]i did not cause significant NO synthesis. We furthermore found that activating adrenoceptors with NE causes synthesis of cGMP by activating a guanylyl cyclase, and that an enhanced [cGMP] evoked by use of caged cGMP causes Ca2+ release from internal stores. Thus, upon sympathetic stimulation, salivary gland acini synthesize NO that, in addition to playing a role in controlling intracellular [Ca2+]i, also might play a role in retrograde signaling processes to the surrounding tissue.

Topics: Acetylcholine; Adrenergic alpha-Agonists; Animals; Calcium; Cells, Cultured; Cyclic GMP; Fluorescein; Guanylate Cyclase; Humans; Indicators and Reagents; Ionomycin; Ionophores; Male; Nitric Oxide; Nitric Oxide Synthase; Norepinephrine; Parasympathomimetics; Parotid Gland; Rats; Rats, Wistar; Receptors, Adrenergic; Salivary Glands; Salivary Glands, Minor; Signal Transduction