Search

This study describes the presence and distribution of cGMP-immunoreactivity and of the nitric oxide (NO) synthesizing enzyme, NO synthase (NOS), as demonstrated by use of the NADPH-diaphorase technique in directly light sensitive pineal organ of the trout. Cyclic GMP immunohistochemistry revealed immunoreactivity in pineal photoreceptor cells that were identified by double-labeling with S-antigen, whereas NADPH-positive structures were located adjacent to these photoreceptor cells. Since NO is known to stimulate synthesis of cGMP, these results indicate a role for NO in pineal function, e.g. in cGMP related events in the phototransduction process as well as in the light-dark control of melatonin synthesis.

Blood-borne angiotensin II (AngII) is known to mediate water-intake by its excitatory effect on neurons in the subfornical organ (SFO). Conversely, nitric oxide (NO) has exclusively inhibitory effects on rat SFO-neurons and on SFO-mediated water-intake. Extracellular and patch-clamp recordings from freshly dissociated rat SFO-neurons showed that glutamate activates AngII-sensitive SFO-neurons by opening ligand-gated cation channels. An immunocytochemical study showed that activation of glutamate receptors increased the concentration of the inhibitory second messenger cGMP in the SFO. A model is proposed suggesting that NO protects SFO-neurons from overexcitability by excitatory neurotransmitters.

The effects of nitric oxide-containing solution and different nitric oxide donors were investigated on spontaneously active neurons using extracellular recording technique in areas of rat spinal cord slices where high levels of nitric oxide synthase are present. In lamina X, 93% of all neurons investigated (n = 84) increased their firing rate and 2% decreased it by superfusion with the nitric oxide donor sodium nitroprusside. In contrast, 49% of all neurons in laminae I and II (n = 90) were inhibited and only 28% were activated. Both effects were due to the postsynaptic action of sodium nitroprusside, because they could still be observed in medium containing 0.3 mM Ca2+ and 9 mM Mg2+, known to block synaptic transmission. Application of 8-bromo-cyclic-GMP caused an excitation of every neuron which was excited by sodium nitroprusside and an inhibition of every cell which was inhibited by sodium nitroprusside (n = 25). This effect was different from the effect of 8-bromo-cyclic-AMP, which mimicked only the excitatory, but not the inhibitory response of sodium nitroprusside. These results provide evidence that nitric oxide in the spinal cord can directly cause an excitation or an inhibition of the electrical activity of spinal neurons. Another, more general conclusion from our results is that the nitric oxide-induced production of cyclic-GMP alone does not allow any prediction about an excitatory or inhibitory effect on the neuronal activity, which has to be determined separately.

The activation of neurons in the subfornical organ (SFO) by angiotensin II (AngII) is well established and is widely regarded as the basis for the AngII-induced increase in water intake. Application of the nitric oxide (NO) donor sodium nitroprusside (SNP) led to an inhibition of the spontaneous electrical activity in 96% of the neurons sensitive for SNP (n = 50). In addition, the firing rate in 60% of the neurons inhibited by SNP decreased in response to superfusion with the natural substrate of the NO synthase (NOS) L-arginine whereas 70% increased their frequency after application of the NOS blocker NG-monomethyl-L-arginine (L-NMMA; n = 10). The inhibitory effect of SNP could be mimicked by application of membrane-permeable 8-Br-cGMP. The presence of nNOS, the neuronal isoform of NOS, was demonstrated immunocytochemically and using the NADPH-diaphorase technique on SFO slices. Using a highly selective antibody against cGMP in formaldehyde-fixed tissue, the NO donors SNP, 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-DL-penicillamine (SNAP) caused a strong increase in cGMP formation when applied under the same conditions as used for the electrophysiological recordings. These electrophysiological results suggest an important role for NO in SFO-mediated responses and offer a plausible explanation for the in vivo-observed opposite effects of AngII and NO on water intake.

Numerous functional studies establish the role of nitric oxide (NO) as a neuromodulator in the central nervous system which affects synaptic transmission. However, there are only a few reports indicating a direct and postsynaptic effect of nitric oxide on the electrical activity of neurons in the central nervous system. The aim of this study was to characterize the effect of nitric oxide on spontaneously active neurons in spinal cord slices using an extracellular recording technique. Because in the lumbar rat spinal cord the NO producing enzyme NO-synthase is primarily located in the superficial dorsal horn (laminae I+II) and around the central canal (lamina X), we restricted our recordings to these areas. While the majority of neurons increased their electrical activity during superfusion with the NO-donor sodium nitroprusside (SNP) in lamina X, neurons in laminae I+II were mainly inhibited by SNP. The excitatory and the inhibitory effects were dose-dependent and reversible and were mimicked by other NO-donors and membrane permeable cyclic guanosine monophosphate (8Br-cGMP) on the same neurons. The spinal cord slice preparation contains functional NO-synthase (NOS), because selective blockade of NOS increased the spontaneous activity of those neurons from laminae I+II which were inhibited by SNP and this effect could be reversed by superfusion with the natural substrate for NOS, L-arginine. It is concluded that NO can activate and inhibit the activity of spinal cord neurons by raising cGMP levels and that these effects are lamina specific. A general consequence of our results is that the NO-induced production of cGMP alone does not allow any prediction about an excitatory or inhibitory effect of NO on the discharge rate of neurons. Thus the NO mediated increase and decrease in neuronal activity is probably the result of intracellular mechanisms downstream from the production of cGMP which results in the activation or inhibition of different ion channels on neurons in laminae I+II and X.

Angiotensin II (ANGII) is known to activate neurons in the subfornical organ (SFO) of mammals and birds and this activation is regarded as the basis for the ANGII induced increase in water intake. Application of the nitric oxide (NO) donor sodium nitroprusside inhibited the activity in 10 out of 12 duck SFO neurons, 8 of which were in addition excited by ANGII. These data, in combination with the histochemical detection of NO synthase in the duck SFO, demonstrate the involvement of NO in SFO mediated responses and might represent the cellular basis for the observed opposite effects of ANGII and NO on water intake.