Evidence showing that beta-endorphin regulates cyclic guanosine 3',5'-monophosphate (cGMP) efflux: anatomical and functional support for an interaction between opiates and nitric oxide

Nitric oxide (NO) is now recognized as a diffusible messenger molecule that normally augments intercellular communication in the central nervous system, but is neurotoxic if released in excessive amounts. NO is synthesized from L-arginine by the Ca2+/calmodulin-dependent neuronal isoform NO synthase (NOS) localized in sub-populations of neurons throughout the brain, including the hypothalamus. In the hypothalamus, NO stimulates the release of GnRH, the primary neurohormone governing reproduction in mammals. Although the excitatory amino acid, glutamate, acting through the N-methyl-D-aspartate (NMDA) receptor is believed to be responsible for stimulation of NO release, the neuronal system(s) that inhibits NO efflux is unknown. As the endogenous opioids, primarily beta-endorphin (betaEND), exert a tonic restraint on GnRH secretion, we sought evidence for a possible functional link between betaEND and NOS pathways in the hypothalamus. We observed that restraining the opioid influence with the opiate receptor antagonist, naloxone, in intact, but not in castrated, rats rapidly augmented extracellular cGMP/NO efflux in the medial preoptic area, where GnRH, NOS, and betaEND immunoreactive pathways are coextensive. Pituitary LH secretion increased in conjunction with this augmented cGMP/NO response and pretreatment with the mu opiate receptor agonist, morphine, suppressed these naloxone-induced responses. Further, visualization of hypothalamic sections immunostained for both betaEND and NOS revealed betaEND-immunoreactive axon terminals in close proximity to NOS-positive cell bodies and dendrites in a number of hypothalamic subdivisions, including the medial preoptic area. These close appositions represented conventional synapses between betaEND nerve terminals and NOS-positive perikarya and dendrites under the electron microscope. Clearly, the experimental data, corroborated by morphological evidence, point to a direct inhibitory control of betaEND on NOS-immunoreactive neurons in monitoring cGMP/NO release. These findings together with the previous observations that the glutamate neurotransmitter acting through NMDA receptors located on NOS-immunopositive cells stimulates cGMP/NO efflux and plasma LH selectively in intact rats document the existence of a dual control comprised of the excitatory NMDA and the inhibitory mu opiate receptors in modulating cGMP/NO release, a response also directed by gonadal steroids. This new knowledge of an inhibitory opioid influence on cGMP/NO release is probably extremely important both in the generation of periodicities in GnRH secretion that underlie hypothalamic control of reproduction and in protecting against neurotoxic overstimulation of NO release by excitatory amino acids.