Isolation of Axonal Varicosities (Autonomic Synaptosomes) From the Enteric Nervous System

Abstract

Abstract— A subcellular fraction enriched with varicosities of autonomic axons was obtained from homogenates of strips of guinea‐pig longitudinal muscle and adherent myenteric plexus using differential and sucrose‐or Ficoll‐density gradient ultracentrifugation. This fraction contained the marker, [G‐3H]5‐hydroxytryptamine (5‐HT), taken up by serotonergic axon terminals present in the preparation during a period of incubation prior to homogenization. Electron microscopic examination showed that the isolated varicosities resembled CNS synaptosomes in containing vesicles and mitochondria but, as is characteristic of autonomic postganglionic terminals, they lacked synaptic membrane specializations. Quantitative electron microscopic radioautography revealed that [G‐3H]5‐HT was confined to isolated varicosities. Isolated varicosities were capable of taking up and sequestering 5‐HT from the surrounding medium; this uptake was temperature‐sensitive and blocked by fluoxetine. The subcellular distribution of [G‐3H]l‐tryptophan was also studied by subfractionation of strips in an attempt to determine which structures were responsible for the high‐affinity uptake of that amino acid. Although most of the [G‐3H]l‐tryptophan was found in the high‐speed supernatant, particulate [G‐3H]l‐tryptophan was most concentrated in the subcellular fraction containing isolated axonal varicosities. These studies indicate that a fraction containing functional serotonergic varicosities can be isolated from the gut. These varicosities are probably one of the elements responsible for the high‐affinity uptake of l‐tryptophan by the myenteric layer of the gut.

Active and inactive pools of nNOS in the nerve terminals in mouse gut: implications for nitrergic neurotransmission

“…The method used for varicosity isolation is summarized in Fig. 1 and was similar to protocols described earlier for varicosities preparation in intestine (10,27). Samples were centrifuged at 1,000 g for 10 min at 4°C to remove undissociated tissue (pellet P1) that was washed once in buffer, the pellet was discarded, and the combined supernatants were further centrifuged at 4,000 g. The pellet P2 obtained after spinning at 4,000 g represented the nuclear fraction and the supernatant was the cytoplasmic fraction.…”

“…Anti-nNOS [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] antibody reactive nNOS. To identify the nNOS bands containing PDZ domain, the Western blots were probed with anti-nNOS [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] antibody (specific for the NH 2 -terminal end of nNOS) that reacts with the PDZ binding site of nNOS.…”

“…To identify the nNOS bands containing PDZ domain, the Western blots were probed with anti-nNOS [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] antibody (specific for the NH 2 -terminal end of nNOS) that reacts with the PDZ binding site of nNOS. The NH 2 -terminal antibody identified 320 and 155-kDa bands on low-temperature SDS-PAGE and only 155-kDa nNOS band on heat-treated SDS-PAGE (Fig.…”

The enteric neural receptor for 5-hydroxytryptamine

“…~8. 25,33. This dual localization would suggest that 5-HT might function as a local hormone and as a neurotransmitter (or neuromodulator).…”

Neuronal and extraneuronal release of ATP and NAD+ in smooth muscle

“…A more direct determination of neuronal release of purines would be to compare release of ATP and NAD + from isolated smooth muscle nerve varicosities in response to depolarizing stimuli. Isolated gastrointestinal nerve varicosities have been used in studies on enteric neurotransmission (53), but simultaneous evaluation of depolarization-induced release of ATP, NAD + and other purines has not been described. Likewise, no data on neurotransmitter storage and release from nerve varicosities or synaptic vesicles in other smooth muscles have been reported.…”