Research highlights fromthe literature

Jens Jordan, MD

j Abstract Recent studies suggestthat genetic variation of thecatecholamine metabolizing en-zyme catechol-O-methyltransfer-ase (COMT) influences painresponsiveness in human sub-jects. Surprisingly, some geneticCOMT variations do not changethe amino acid sequence. Instead,the secondary messenger RNA isaltered such that more or lessprotein is produced. Animal stud-ies by the same group showedthat pharmacological COMT inhi-bition increases pain responsive-ness through beta-2 and beta-3adrenoreceptor stimulation. An-other publication sheds new lighton the predictive value of auto-nomic testing in young patientswith type 1 diabetes mellitus.Cardiovascular autonomic reflextesting did not predict occurrenceof early nephropathy, retinopathy,or hypoglycemia unawarenessover a twelve years period. Yet,impaired autonomic pupillaryfunction was associated with amarkedly increased risk for bothnephropathy and retinopathy.The study might suggest thatautonomic neuropathy precedesmicrovascular disease. Finally,another study investigated neuralcircuits contributing to centralcommand responses that occurwith arousal and physical exer-cise. Cholinergic projections fromthe pedunculopontine tegmentalnucleus appear to activate mus-carinic cholinergic receptors onrostral ventrolateral medulla neu-rons. Sympathetic activation thenensues.

j Key words catechol-o-methyl-transferase Æ COMT Æ diabetesmellitus Æ diabetic neuropathy Æcentral command Æ pedunculo-pontine tegmental nucleus Ærostral ventrolateral medulla

Is catechol-O-methyltransferaseon your radar?

Released norepinephrine is largelyreclaimed by postganglionicadrenergic neurons and repack-aged or metabolized by mono-amine oxidase. A smallerpercentage escapes the synapseand may enter the systemic cir-culation. This proportion of nor-epinephrine is taken up byextraneuronal tissue and mainlymetabolized by catechol-O-meth-yltransferase (COMT). COMT ishighly expressed in liver and kid-ney. The human COMT gene en-codes two different proteins,namely soluble and membranebound COMT. Several studiessuggested association of commonCOMT gene polymorphisms withpsychiatric and neurological dis-orders. Given COMT’s relativelysmall contribution to peripheralnorepinephrine metabolism, theenzyme has been somewhat ne-glected by autonomic scientists.Recent data on COMT and painsensitivity may renew the interestin its physiology.

Diatchenko et al. [1] tested howgenetic variations in the COMTgene influence pain sensitivity inhuman subjects. Genetic studiesare complicated by the fact thatmany genes contain large numbersof polymorphic sites. The theo-retical number of gene variants(i.e., haplotypes) is large. Not all ofthese predicted haplotypes occurin real life as many polymor-phisms are inherited together.Therefore, the authors examined

the association between pain re-sponses and certain COMT hapl-otypes rather than individualpolymorphisms. COMT haplo-types associated with low (LPS),average (APS), and high (HPS)pain sensitivity had been identi-fied previously. In any event, LPS/LPS homozygotes had the lowestand APS/HPS heterozygotes hadthe greatest pain responsiveness.Furthermore, the authors showedassociation between one poly-morphisms leading to substitutionof valine by methionine in posi-tion 158 and a specific pain qual-ity, namely temporal summationof heat pain. The amino acidsubstitution reduces COMT’sthermostability and enzymaticactivity. Together, the data suggestthat COMT gene variations leadingto decreased enzyme activity areassociated with increased painsensitivity.

Nackley et al. [2] studied howthe previously mentioned LPS,APS, and HPS COMT haplotypesalter enzyme activity. Remarkably,both, LPS and HPS contain valinein position 158. The fact that thenucleic acid variation characteriz-ing LPS and HPS do not lead toamino acid changes is confusing.First, the authors predicted themRNA structure for each haplo-type using specialized software.The predicted mRNA structurediffered markedly between LPS,APS, and HPS. Second, the authorsproduced complementary DNA’sfor soluble and membrane boundCOMT that differed only in thethree nucleic acids correspondingto LPS, APS, and HPS haplotypes.Third, they introduced (i.e.,transfected) the complementaryDNA’s in mammalian cells. Fi-nally, the authors quantifiedCOMT mRNA and protein levelsas well as COMT activity in thesecells. Compared with LPS trans-fected cells, COMT protein

AUTONOMIC NEWSClin Auton Res (2007) 17:2–4DOI 10.1007/s10286-007-0398-z

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expression and COMT expressionwas much reduced with HPStransfected cells. Surprisingly, thedifference was not related tomRNA abundance or degradation.Further experiments suggestedthat the structure of the messengerRNA affected production ofCOMT protein. The observationhas important implications be-yond COMT genetics. The studysuggests a mechanism by whichDNA polymorphisms that do notlead to an amino acid exchangenonetheless profoundly alter pro-duction of the respective protein.

In yet another study, Nackleyet al. [3] tested how reduced cat-echolamine degradation throughCOMT could increase pain sensi-tivity. The authors conductedstandardized pain tests in rats inthe presence and in the absence ofpharmacological COMT inhibi-tion. They applied different COMTinhibitors to exclude a non spe-cific response. To determinemechanical pain responsiveness,the authors applied a filament to apaw. Thermal pain responsivenesswas assessed by placing a paw on aheated plate. In both sets ofexperiments the authors observedhow much of each stimulus ittakes until rats withdrew the paw.

Pharmacological COMT inhibi-tion increased pain responsivenessregardless of the stimulus. Toevaluate the degree of painresponsiveness elicited by COMTinhibition, the authors also testeda classical stimulus. In this test,carrageenan is injected in a hindpaw. The injection induces localinflammation, thereby increasingpain sensitivity. Remarkably, hy-peralgesia caused by COMT inhi-bition was similar to carrageenan-induced hyperalgesia. In sub-sequent experiments, the authorstested the hypothesis that the in-crease in pain responsivenessthrough COMT inhibition is ex-plained by beta-adrenoreceptorstimulation. Indeed, the responsewas abolished with non selective

beta-adrenoreceptor blockade butnot with alpha-adrenoreceptor ordopamine receptor blockade. Theauthors showed that beta-2 andbeta-3 adrenoreceptors are in-volved. Together, these studiessuggest that COMT may have agreater effect on adrenergic re-sponses in peripheral tissues thatpreviously believed. COMT de-serves more of our attention.

To do or not to do - autonomictesting in patients with diabe-tes mellitus

Ewing’s landmark studies in the1970’s suggested that cardiacautonomic neuropathy is associ-ated with increased mortality inpatients with type 1 diabetesmellitus. The increased mortalityis at least in part explained bycardiovascular complications.Furthermore, autonomic neurop-athy may increase the risk ofhypoglycemia unawareness. Theseobservations created much enthu-siasm amongst autonomically ori-ented physicians. Many patientswere submitted to autonomicnervous system testing. Was allthis effort worth the money or arethere cheaper tests that give simi-lar prognostic information? Forexample, measurement of albuminexcretion in urine is much lessexpensive and time consumingthan autonomic testing.

The study by Maguire et al. [4]addressed the issue in young pa-tients with type 1 diabetes mell-itus. The authors included 335adolescent patients in their study.Of those, 137 patients consented toparticipate in a follow up study. Inthis group, diabetes duration be-fore the baseline evaluation was6.3 years. An average HbA1c levelof 8.2% at baseline suggests poorglycemic control in many partici-pants. Patients underwent cardio-vascular autonomic reflex testingincluding determination of respi-ratory sinus arrhythmia, a Val-

salva maneuver, and orthostatictesting during the baseline evalu-ation. Furthermore, the investiga-tors assessed autonomic control ofpupils using computerized pupill-ometry. Earlier cross sectionalstudies suggested that abnormali-ties in pupillary function mayprecede renal damage. After afollow up period of approximatelytwelve years, patients were exam-ined for nephropathy, retinopathy,and hypoglycemia unawareness.Early diabetic nephropathy wasdetected by measuring urinaryalbumin excretion. Retinopathyand hypoglycemia unawarenesswere diagnosed with stereoscopicfundus photography and a vali-dated questionnaire, respectively.

Glycemic control had improvedslightly towards the end of thefollow up period. An increase inurinary albumin was shown in19% of the patients. None devel-oped severe renal failure duringfollow up. Retinopathy was ob-served in 69% of the patients. Only55% were completely aware of allhypoglycemic episodes. Baselinecardiovascular autonomic testingdid not predict the occurrence ofmicroalbuminuria or retinopathy.However, in patients with restingpupillary diameter at baseline inthe lower quartile (<4.7 mm), therisk to develop microalbuminuriaor retinopathy was increased 4-5fold. Neither cardiovascular auto-nomic testing nor pupillometrypredicted the risk for hypoglyce-mia unawareness.

The important finding of thestudy by Maguire et al. [4] is thatautonomic neuropathy as evi-denced by altered pupillary sizemay precede microvascular dis-ease in young patients with type 1diabetes mellitus. Owing to dif-ferences in the cardiovascular andmetabolic risk profile, these find-ings cannot be simply extrapolatedto older patients with type 2 dia-betes. The clinical implications arenot straightforward. Clearly, pa-tients with symptomatic auto-

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nomic neuropathy require auto-nomic testing to guide therapy.However, still unclear is whetheror not autonomic testing shouldbe considered a screening tool inasymptomatic patients. Does itinfluence therapeutic decisionsand more importantly does it im-prove prognosis? No one knows,but further studies in this areashould be done.

Central command, who is incharge?

During arousal and physical exer-cise, the brain can activate thesympathetic nervous system in afeed-forward fashion. The mecha-nism is often called ‘‘central com-mand’’. The increase insympathetic activity raises heartrate and blood pressure even beforephysical exercise, which ensuressufficient organ perfusion. How-ever, the ‘‘fight and flight’’ responseis often maladaptive in a modernenvironment. Diaphoresis andheart pounding do not help muchwhen you try delivering a talk orsweat through an oral examination.Remarkably, the neural circuitryregulating central command re-sponses is poorly understood.

Earlier studies suggested thatmuscarinic cholinergic receptorsin the brain regulate blood pres-sure, body temperature, and res-piration. Neural connectionsbetween midbrain and medullacould be involved. Therefore,Padley et al. [5] tested thehypothesis that cholinergic pro-jections from the pedunculopon-tine tegmental nucleus (PPT) in

the midbrain to the rostral ven-trolateral medulla (RVLM) medi-ate central command likeresponses. The PPT is componentof the reticular activating systemthat influences arousal and moti-vation while the RVLM containsneurons generating basal sympa-thetic tone.

The authors conducted theirstudies in anesthetized rats.Among other measurements, theyrecorded blood pressure, heartrate, and splanchnic sympatheticnerve activity. Firstly, they showedthat activation of muscarinic ace-tylcholine receptors raised sym-pathetic activity and bloodpressure. The sympathetic baro-reflex curve was reset to higherblood pressure values. All theseresponses were attenuated whenmuscarinic receptors were locallyblocked in the RVLM. Then, theauthors demonstrated that cho-linergic neurons are located in thevicinity of RVLM neurons. RVLMneurons expressed muscarinicacetylcholine receptors of the M2and M3 subtype. Retrograde tracerexperiments showed cholinergicprojections from the PPT to theRVLM. Chemical stimulation ofPPT neurons elicited a pressorresponse through sympatheticactivation and facilitated thesympathetic baroreflex. Musca-rinic cholinergic receptor block-ade attenuated the change inbaroreflex function.

The study suggests that cho-linergic neurons originating in thePPT and traveling to the RVLMmay be an important componentof central command pathways.Many questions remain. Yet, Pad-

ley et al. [5] helped to turn ‘‘cen-tral command’’ from an almostphilosophical term into a well-de-fined physiological concept. Thestudy may have clinical implica-tions because many medicationsalter cholinergic transmission inthe brain.

References

1. Diatchenko L, Nackley AG, Slade GD,et al. (2006) Catechol-O-methyltransfer-ase gene polymorphisms are associatedwith multiple pain-evoking stimuli. Pain125(3):216–224

2. Nackley AG, Shabalina SA, Tchivileva IE,et al. (2006) Human catechol-O-methyl-transferase haplotypes modulate proteinexpression by altering mRNA secondarystructure. Science 314(5807):1930–1933

3. Nackley AG, Tan KS, Fecho K, Flood P,Diatchenko L, Maixner W (2006) Cate-chol-O-methyltransferase inhibition in-creases pain sensitivity throughactivation of both beta(2)- and beta(3)-adrenergic receptors. Pain (in press)

4. Maguire AM, Craig ME, Craighead A,et al. (2007) Autonomic Nerve TestingPredicts the Development of Complica-tions: A 12-year follow-up study. Dia-betes Care 30(1):77–82

5. Padley JR, Kumar NN, Li Q, Nguyen TB,Pilowsky PM, Goodchild AK (2007)Central Command Regulation of Circu-latory Function Mediated by DescendingPontine Cholinergic Inputs to Sympat-hoexcitatory Rostral Ventrolateral Me-dulla Neurons. Circ Res (in press)

J. Jordan, MD (&)Franz Volhard Clinical Research CenterMax Delbruck Center, andHELIOS Klinikum BerlinCharitu Campus Buch, Haus 129Wiltbergstr. 5013125 Berlin, GermanyTel.: +49-30/9417-2220Fax: +49-30/9417-2265E-Mail: jens.jordan@charite.de

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