Reply to R. Weinberg and H. Schmidt

402 evidence supports current anatomical methods as sensitive probes for NOS at sites of potential NO synthesis.

References Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhi, M., Dawson, T.M. and Snyder, S.H., Nitric oxide synthase protein and mRNA arc discretely localized in neuronal population of mammalian central nervous system together with NADPH diaphorase, Neuron. 7 (1991) 61.5-624. Courtoy, P.J., Picton, D.H. and Farquhar, M.G., Resolution and limitations of the immunoperoxidase procedure in the localization of extracellular matrix antigens, J. Histochem. Cytochem., 31 (19831945-951. Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H., Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 88 (1991) 7797-7801. Dun, N.J., Dun. S.L., Fiirstermann, U. and Tseng, L.F., Nitric oxide synthase immunoreactjvi~ in rat spinal cord, Neurosci. Lett., 146 (19921217-220. Hiki, K., Hattori, R., Kawai, C. and Yui, Y., Purification of insoluble nitric oxide synthase from rat cerebellum, J. Biochem., 111 (1992) 556-558. Hope, B.T., Michael, G.J., Knigge, KM. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Nat]. Acad. Sci. USA, 88 (199112811-2814. Janssens, SP, Shimouchi, A., Quertermous, T., Bloch, D.B. and Bloch, K.D., Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase, J. Biol. Chem., 267 (1992) 14519-14522. Klatt, P., Heinzel, B., John, M., Kastner, M., Bohme, E. and Mayer, B., Ca*+/calmodulin-de~ndent cytochrome C reductase activity of brain nitric oxide synthase, J. Biol. Chem., 267 (1992) 1127411248. Llewellyn-Smith, I.J., Song, Z.-M., Costa, M., Bredt, D.S. and Snyder, S.H., Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons, Brain Res., 577 (1992) 337-342. Mitchell, J.A., Kohlhaas, K.L.. Matsumoto, T., Potlock, J.S., Forstermann, U., Warner, T.D., Schmidt, H.H.H.W. and Murad, F.. Induction of NADPH-dependent diaphorase and nitric oxide synthase activity in aortic smooth muscle and cultured macrophages, Mol. Pharmacol., 41 (1992) 1163-1168. Meller, S.T. and Gebhart, G.F., Nitric oxide (NO) and nociceptive processing in the spinal cord, Pain, 52 (1993) 127-136. Messing, A., Stieber, A. and Gonatas, N.K., Resolution of diamino~nzidine for the detection of horseradish peroxidase on surfaces of cultured cells. J. Histochem. Cytochem.. 33 (19851 837-839. Pollock, J.S., Fdrstermann, U., Mitchell, J,.A., Warner, T.D., Schmidt. H.H.H.W., Nakane, M. and Murad, F., Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells, Proc. Natl. Acad. Sci. USA, 88 (1991) 10480-10484. Saffrey, M.J., Hassall, C.J., Hoyle, C.H., Belai, A. Moss, J., Schmidt, H.H.H.W., Forstermann, U., Murad, F. and Burnstock, G., Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones, NeuroReport, 3 (1992) 333-336. Schmidt, H.H.H.W., Smith, R.M., Nakane, M. and Murad, F.. Ca’+/caImodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca’+/caImoduIin-independent diaphorase and reductase activities, Biochemistry, 31 f1992a) 3243-3249. Schmidt, H.H.H.W., Gagne, G.D., Nakane, M., Pollock, J.S., Miller, M.F. and Murad, F., Mapping of neural nitric oxide synthase in the rat suggests frequent collocation with NADPH diaphorase

but not soluble guanylyi cyclase and novel paraneural functions for nitrinergic signal transduction, J. Histochem. Cytochem., 40 (1992b) 1439-1456. Schmidt, H.H.H.W., Warner. T.D., Nakane, M. Fiirstermann, II. and Murad, F., Regulation and subcellular location of nitrogen oxide synthases in RAW 264.7 macrophages, Mol. Pharmacol., 41 (1992~1, 615-624. Valtschanoff, J., Weinberg, R.J. and Rustioni, A.. NADPH diaphorase in the spinal cord of rats, J. Comp. Neural., 321 (1992a) 209-222. Valtschanoff, J., Weinberg, R.J., Rustioni, A. and Schmidt, H.H.H.W., Nitric oxide synthase and GABA colocalize in lamina II of rat spinal cord, Neurosci. Lett., 148 (1992bl6-10. Valtschanoff, J., Weinberg, R.J., Kharazia, V.N., Nakane M. and Schmidt, H.H.H.W. Neurons in rat hippocampus that synthesize nitric oxide, J. Comp. Neural., 331 (1993af 111-121. Valtschanoff, J., Weinberg, R.J., Kharazia, V.N., Schmidt, H.H.H.W., Nakane, M. and Rustioni, A., Neurons in rat cerebral cortex that synthesize nitric oxide: NADPH diaphorase histochemistry, NOS immun~yt~hemist~, and colocaiization with GABA, Neurosci. Lett., 157 (1993b) 157- 161. Wolf, G., Wiirdig, S. and Schiinzel, G., Nitric oxide synthase in rat brain is predominantly located at neuronal endoplasmic reticulum: an electron microscopic demonstration of NADPH-diaphorase activity, Neurosci. Lett., 147 (1992) 63-66. Young, H.M., Furness, J.B., Shuttleworth, C.W.R., Bred& D.S. and Snyder, S.H., Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine, Histochemistry, 97 (1992) 375-378. Richard J. Weinberg L&X of CellBiology and Uniwrsity ofNorth

Anatomy Carolina, Chapel Hi& NC 27599, USA

Harald H.H.W. Schmidt Ikpi.

of Clinical

Biochemistry and Pathobiochemiwy Medical Unicvrsity Clinic Wiirzhurg Yersbacher Str. 5

PAIN 02462

Reply to R. Weinberg and H. Schmidt We would like to thank Drs. Weinberg and Schmidt for their comments on our recent review in Pain. We have no disagreement with them that the biochemicaf evidence they provide is compatible with the suggestion that the constitut~e brain enzyme nitric oxide sythase (NOS) is an NADPH-de~ndent diaphorase. However, there are many different diaphorases that are NADPH-dependent. One of the major confounding factors in the literature has been the suggestion that the NADPH-diaphorase which survives aldehyde fixation is equivalent to brain NOS. However, there is no evidence of which we are aware that has addressed the question of just what is the unique structural feature of this NADPH diaphorase that prevents aldehyde degradation. This evidence is crucial to the argument that the enzymes are the same. Certainly, it has been shown that NADPHdependent cytochrome P450 reductase has a similar structural homology to brain NOS (Bredt et al. 1991). Both enzymes are similar in that they have NADPH, FAD and FMN binding sites; however,

403 cytochrome P450 reductase does not survive aldehyde fiiation, emphasizing our concern. Recently, Matsumoto et al. (1993) have questioned the validity of the claim that NADPH-diaphorase is equivalent to NOS. In that study they examined the effect of different concentrations of formaldehyde fixation on NADPH-diaphorase and NOS. They suggest that the correlations between the two may be coincidental. In their study they showed a clear lack of correlation between brain regions containing NOS and NADPH-diaphorase and suggested that NADPH-diaphorase activity, unrelated to NOS, may survive different fixation protocols. This has not been systematically investigated. In addition, it has not been shown that what remains after aldehyde fixation is an enzyme and not some breakdown product that is able to utilize NADPH. Further, and most importantly, there is no published immunocytochemical evidence of which we are aware which shows that NOS and NADPH-diaphorase are localized within the same neuron in the spinal cord (this may be the case in the peripheral nervous system, but it has not been shown in the brain stem and spinal cord). Indeed, Dun et al. (1992) have reported a lack of correlation between NOS immunoreactivity and NADPH-diaphorase localization and evidence from our laboratory (Meller et al. unpublished observations) and from the laboratories of Dr. AI Beitz in Minnesota and Dr. Thomas Herdegen in Heidelberg indicate that there is certainly less than 100% homology between the two in the brain stem and spinal cord. Further, it is important to stress that only quantitative double-labeling studies will definitively answer the colocalization question - qualitative descriptions or analysis of adjacent sections is not sufficient. Accordingly, while the majority of the biochemical data indicates that the enzymes are the same, there is no immunocytochemical evidence to support this. To the contrary, there is evidence, unpublished at the present time, that these enzymes do not colocalize in the spinal cord or brain stem. This issue will likely be resolved in the near future. We thank Drs. Weinberg and Schmidt for raising this issue and permitting this discussion.

Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed, R.R. and Snyder, S.H., Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature 351 (1991) 714-718. Dun, N.J., Dun, S.L., Forstermann, U. and Tseng, L.F., Nitric oxide synthase immunoreactivity in rats spinal cord, Neurosci. Lett., 147 (1993) 217-220. Matsumoto, T., Nakane, M., Pollock, J.S., Kuk, J.E. and Forstermann, U., Correlation between soluble nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue to fiiative, Neurosci. L&t., 155 (1993) 61-64.

tial tissue damage or expressed in terms of such damage”. With such a definition the investigation of pain presupposes a communication between a person in pain and an observer. A secure verbal communication, lacking misinterpretations, can be difficult to achieve even between two persons and is, of course, utopic between humans and other species. Still, in numerous papers reporting data from animal studies on mechanisms probably related to pain, words such as pain, hyperalgesia and allodynia are used as well as phrases such as “pain-like behaviour”, “. . . produces disorders of pain sensation like those seen in man”, “. pain-related syndrome” or “. abnormal pain sensations”. This terminology can, in our view and in conformity with the IASP definition, only relate to humans and is not valid in animal studies and should therefore be excluded in reports on animal research. It is impossible and naive to extrapolate from behavioural studies in animals to the complex sensation of pain in humans regardless of if, e.g., significant correlations between electrophysiological recordings and behaviour can be demonstrated in animal studies. Furthermore, the use of an identical nomenclature in human and animal research can give the false impression to laymen and others, without expert knowledge but involved in research financing and health care work, of a secure and unrestricted parallelism between species in all aspects. In addition, we would like to emphasize that treatment strategies effective in attenuating different behaviours in animals, behaviours thought to relate to pain, has proven to be totally without pain relieving potency in humans with a presumed similar condition. One way to solve the issue we are pursuing is to create a specific terminology for what is being observed in animal experiments. One example is the term autotomy adopted to describe the biting, scratching and self-mutilating behaviour of a rat with a partially denervated extremity after experimental nerve injury. Autotomy could thus be defined without any contamination with words relating to communicated sensations in humans, thus avoiding associating the behaviour with, e.g., neurogenic pain as seen in humans. As a further example instead of using the words allodynia and hyperalgesia in models of mononeuropathy one can use phrases such as “decreased latency to withdrawal reactions following tactile and nociceptive stimuli, respectively”. We feel that it is timely to discuss the relevance and consequences of a similar terminology used in human and animal studies aiming at exploring the field of pain. We suggest that a separate taxonomy should be developed, preferably by the IASP task force on taxonomy, to characterize presumed pain related phenomena in animals. Such a rationale would put data from animal research in a sound perspective, i.e., extrapolations from animal data offer possible implications only, for the understanding of pain in humans. There is a need for both human and animal research to unravel the various aspects of pain. In the end, however, it will be studies on humans in pain only that can give us the final answers.

Stephen T. Meller G.F. Gebhart

Anders Ekblom

References

of Pharmacology University ofIowa Iowa City, IA 52242, USA

Department

Department

ofAnaesthesiology

and Intensive Care Karolinska Hospital Stockholm, Sweden

Per Hansson PAIN 02464 Department

When is “pain”

appropriate?

According to the definition of pain adopted by the IASP pain “is a subjective and emotional experience associated with actual or poten-

Neurogenic Pain Unit of Rehabilitation Medicine Karolinska Hospital Stockholm, Sweden