Further characterisation of particulate neuronal nitric oxide synthase in rat small intestine.

Neuronal NO-synthase (nNOS) was investigated in rat longitudinal muscle/myenteric plexus (LM/MP) tissue at the cellular and subcellular level. Using preparations and double immune staining and light and electron microscopy, we concluded that, in these preparations, nNOS is only present in neuronal cells. However, in spite of numerous attempts to morphologically identify the NOS-containing subcellular structure, no firm conclusions were possible. Consequently, the problem was approached by biochemical methods including gradient centrifugation followed by analysis of the fractions. Using a protocol involving gentle homogenisation of the tissue, we found that about 10% of the nNOS immune reactivity was particle-bound confirming previous results (Biochem. Pharmacol. 60 (2000) 145). However, applying a different protocol including strong homogenisation, we now demonstrated that about 50% of the immune reactive nNOS was sedimentable. The results suggested that particulate nNOS is associated with one single subcellular structure, which is different from the plasma membrane, rough and smooth endoplasmic reticulum, mitochondria and lysosomes. The equilibrium sedimentation characteristics of the nNOS containing particles corresponded partly to those containing vasoactive intestinal polypeptide (VIP) or synaptobrevin. Application of non-equilibrium centrifugation conditions, however, demonstrated that almost no co-localisation occurred. We conclude that, in the LM/MP tissue, nNOS is about 50% particle-bound in a subcellular structure, which is different from the VIP-containing particle and from synaptobrevin-containing exocytotic particles.

Cellular activity of dopamine DA2 receptor splice variants and mutants.

Two splice variants of the dopamine D2 (DA2) receptors-a long (DA2l) and short (DA2s) form-and two corresponding mutants (serine at position 311 replaced by a cysteine) have been described. Using CHO-cells transfected with the genes for the splice variants and their respective mutants and a bioassay based on the online registration of the extracellular acidification rate (ECAR) of intact cells, we investigated the cellular activity upon stimulation of the receptor. We first confirmed that the acute response upon short agonist stimulation was significantly higher for DA2s than for DA2l. However, in contrast to the ongoing opinion, we found that the desensitisation pattern upon long-term agonist treatment was indistinguishable for both wild-type receptors. As far as the corresponding ser311cys mutated receptors are concerned, the concentration-response curves and the desensitisation pattern were superimposable to the corresponding wild-type receptors. Inhibition of protein kinase C (PKC) had very little effect both on the concentration-response curves and on the desensitisation pattern. We conclude that signal transduction following stimulation of DA2 receptors in the CHO expression system is more effective for DA2s than for DA2l. The point mutation in position 311 has little impact on the downstream signalling of DA2 receptors.